Retinoid X Receptor Ligand Research Articles

SREBP-1c mediates the retinoid-dependent increase in fatty acid synthase promoter activity in HepG2

Treatment of HepG2 with all- trans retinoic acid (RA) induces expression of fatty acid synthase (FAS) mRNA and protein. Transfections show that the FAS promoter positively responds to retinoid X receptor (RXR) but not to RA receptor (RAR) agonists. Since RXR alone is capable of mediating the RA response of FAS, the existence of a classical RA-responsive element in the FAS promoter may be ruled out. Binding sites for NF-Y and SREBP-1 proved to be essential for the RA response. Exposure to all- trans RA increased mRNA and protein levels of SREBP-1, a transcriptional activator for FAS. Overexpression of a dominant-negative form of SREBP-1c diminished the RA-dependent increase in promoter activity. These data demonstrate that RXR ligands can stimulate the expression of a lipogenic gene solely by inducing transcription and cleavage of membrane-bound SREBP-1c.

Thermodynamic Characterization of the Interaction between CAR−RXR and SRC-1 Peptide by Isothermal Titration Calorimetry

The constitutive androstane receptor (CAR) enhances transcription of specific target genes that regulate several metabolic pathways. CAR functions as an obligate heterodimer (CAR-RXR) with the retinoid X receptor (RXR). Also part of the active receptor complex is the steroid receptor coactivator-1 (SRC-1) which interacts with the receptor complex via specific receptor interaction domains (RIDs). A peptide derived from SRC-1 RID2 is used to study the thermodynamic properties of the interaction with the CAR-RXR ligand binding domain (LBD) complex. In the absence of ligands for both CAR and RXR, binding of coactivator peptide to the CAR-RXR heterodimer is characterized by a favorable enthalpy change and an unfavorable entropy change. The addition of the CAR agonist, TCPOBOP, increases the affinity for coactivator by decreasing the unfavorable entropy and increasing the favorable intrinsic enthalpy of the interaction. The RXR ligand, 9-cis-RA, generates a second SRC-1 site and increases the affinity by improving the entropic component of binding. There is an additional increase in affinity for one of the two sites in the presence of both ligands. The change in heat capacity (deltaCp) is also investigated. A 2-fold difference in deltaCp is observed between liganded and unliganded CAR-RXR. The observed thermodynamic parameters for binding of SRC-1 peptide to liganded and apo CAR-RXR as well as the difference in the deltaCp data provide evidence that the apo CAR-RXR heterodimer is conformationally mobile. The more favorable enthalpic contribution for TCPOBOP-bound CAR-RXR indicates that preformation of the binding site improves the complementarity of the coactivator-receptor interaction.

Targeting Nur77 translocation

The ultimate growth of a tumour depends on not only the rate of tumour cell proliferation, but also the rate of tumour cell death (apoptosis). Nur77 (also known as TR3 or NGFI-B), an orphan member of the nuclear receptor superfamily, controls both survival and death of cancer cells. A wealth of recent experimental data demonstrates that the Nur77 activities are regulated through its subcellular localisation. In the nucleus, Nur77 functions as an oncogenic survival factor, promoting cancer cell growth. In contrast, it is a potent killer when migrating to mitochondria, where it binds to Bcl-2 and converts its survival phenotype, triggering cytochrome c release and apoptosis. Agents, such as 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437), which induce Nur77 migration from the nucleus to mitochondria, effectively induce apoptosis of cancer cells. Moreover, Nur77 translocation is highly controlled by retinoid X receptor (RXR), suggesting a role of RXR ligands in regulating the process. Thus, translocation of Nur77 from the nucleus to mitochondria represents a new paradigm in cancer cell apoptosis, and targeting the Nur77 translocation by AHPN/CD437 or RXR ligands promises to effectively restrict cancer cell growth by simultaneously promoting cancer cell death and suppressing cancer cell proliferation.

The Combination of 9-cis Retinoic Acid Plus MEK Inhibitor Induces Apoptosis Via Dephosphorylation of Retinoid X Receptor α in HL-60R Cells.

Resistance against retinoic acid (RA) is a serious problem of differentiation-induction therapy for acute promyelocytic leukemia (APL) in clinical practice. RA exerts its biological activities primarily through the nuclear receptor dimmer, consisting of retinoic acid receptor (RAR) and retinoid X receptor (RXR). Both receptors consist of three subtypes (α, β and γ) and form heterodimeric RAR/RXR and homodimeric RXR/RXR complexes. 9-cis RA, which is a high-affinity ligand for RXR but also binds to RAR, induces cell differentiation in wild type HL-60 human myeloid leukemia cells. However, in HL-60R cells, the RA-resistant subclone of HL-60, 9-cis RA can not induce cell differentiation and apoptosis. We recently reported that malfunction of RXRα due to posttranslational modification by phosphorylation to be associated with carcinogenesis of hepatocellular carcinoma (HCC). Phosphorylated form of RXRα (p-RXRα) at serine 260 by Ras/MAPK is resistant to ubiquitin/proteasome-mediated degradation and the accumulation of p-RXRα interferes with the function of remaining normal RXRα in a dominant negative manner, thereby promoting growth of HCC cells. We also found that in the presence of MEK inhibitor PD98059, 9-cis RA can induce the degradation of p-RXRα and thus restoring the function of this receptor in RXRα-phosphorylated human HCC cells. Based on the results as described above, we initiated this study to examine whether 9-cis RA can exert growth inhibitory effects on RA-resistant HL-60R cells when combined with MEK inhibitor, with focusing on the inhibition of expression of p-RXRα protein. We found that RXRα protein was originally expressed in both HL-60 and HL-60R cells, and that the expression level of RXRα protein was inhibited by about 60% and 20%, respectively, when those cells were treated with 0.5 μM 9-cis RA. Not only total RXRα protein, but also the level of p-RXRα protein was constitutively expressed in both HL-60 and HL-60R cells, and was significantly decreased in HL-60 cells by treatment with 9-cis RA alone. On the other hand, in HL-60R cells, 9-cis RA alone nor 20 μM PD98059 alone did not cause a down regulation of these proteins. However, when HL-60R cells were treated with the combination of 9-cis RA plus PD98059, the expression level of p-RXRα protein was markedly decreased. Moreover, the combination of these agents induced apoptosis in HL-60R cells, whereas similar effect was not obtained when the cells were treated with either agent alone. The combined treatment of these agents also cooperatively inhibited the growth of HL-60 R cells. The present findings suggest that the accumulation of p-RXRα might impair the function of normal RXRα as a master regulator of nuclear receptors and, thus, contributing to the resistance to RA in HL-60R cells. Combination of 9-cis RA plus MEK inhibitor might be an effective regimen for patients with RA resistant APL.

Cell Surface Localization of ABCG1 Does Not Require LXR Activation

To the Editor: In a recent article in this journal entitled “LXR-Induced Redistribution of ABCG1 to Plasma Membrane in Macrophages Enhances Cholesterol Mass Efflux to HDL”, Nan Wang and colleagues showed that in nonstimulated macrophages ABCG1 protein is predominantly intracellular, and that on treatment with a synthetic liver X-receptor (LXR) ligand it increased in amount and appeared to relocate from this intracellular pool to the plasma membrane.1 Based on our findings outlined below, we suggest an alternative interpretation of these results. Experiments performed in our laboratory showed that transiently expressed N-terminally EGFP-tagged ABCG1 fusion protein localized in the Golgi apparatus and in intracellular vesicles of epithelial HeLa cells a few hours after transfection. Then, between 6 and 24 hours after transfection, increasing amounts of EGFP-ABCG1 appeared in the plasma …

The retinoid X receptor ligand restores defective signalling by the vitamin D receptor

It is assumed that the retinoid X receptor (RXR) acts as a silent partner to the vitamin D receptor (VDR) with its only function to increase affinity of VDR/RXR to its DNA recognition site. In this study, we show that the RXR ligand 9-cis-retinoic acid (9-cis-RA) induces recruitment of coactivators by the DNA-bound heterodimer and potentiates vitamin D-dependent transcriptional responses. The presence of 9-cis-RA increases induction of cyp24 transcripts and differentiation of colon cancer cells by vitamin D, confers significant agonistic activity to a VDR ligand with very low agonistic activity and can even restore transcriptional activity of an AF-2 mutant VDR that causes hereditary rickets. This study shows that, in VDR/RXR heterodimers, allosteric communication triggered by the RXR ligand has a previously unrecognized role in vitamin D signalling, with important physiological and therapeutic implications.

Fetal Alcohol Syndrome and Essential Fatty Acids

Essential fatty acids (EFAs) are structural components of the brain and influence nerve conduction and transmitter release and action [ 1]. Prostaglandins (PGs) derived from EFAs and polyunsaturated fatty acids (PUFAs) modify nerve conduction and transmitter function. Ethanol (1) reduces blood linoleic acid (LA) levels; (2) blocks conversion of LA and gamma-linolenic acid (GLA) to arachidonic acid (AA), as well as alpha-linolenic acid (ALA) to eicosapentaenoic and docosahexaenoic acids (EPA and DHA, respectively), by inhibiting delta-6 and delta-5 desaturases; and (3) enhances the conversion of dihomo-gamma-linolenic acid (DGLA) to PGE1 [ 1]. Ethanol-enhanced interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) production suggest that immunological mechanisms play a role in ethanol-induced diseases. Cerebral cortex from chronic ethanol-fed rats showed up-regulation of inducible nitric oxide (iNOS), cyclo-oxygenase-2 (COX-2), IL-1-beta, and activation of transcription factors nuclear factor-kappa B (NF-kappa B) and AP-1—effects that increased both caspase-3 and apoptosis [ 2]. Brains of ethanol-treated mice show raised phospholipase-A2 and phospholipase-C activity [ 3] that can cause the release of AA, EPA, and DHA from membrane phospholipids. AA, EPA, and DHA form precursors to eicosanoids—lipoxins (LXs), resolvins, and neuroprotectin D1 (NPD1) [ 4]. LXs, resolvins, and NPD1 suppress IL-6 and TNF-alpha production. PGs, LXs, resolvins, and NPD1 precursor supplies are limited; therefore, when ethanol consumption is substantial, tissue concentrations of PUFAs decline, leading to a fall in the synthesis of various PGs, LXs, resolvins, and NPD1. DHA and other PUFAs released from the membrane in response to neurotransmitters [ 5] activate retinoid X receptor (RXR) as PUFAs form ligands for the RXR [ 6]. DHA deficiency results in impaired spatial learning and other abnormalities [ 7]. RXR heterodimerization partners peroxisome proliferator-activated receptors (PPARs), liver X receptors, and farnesoid X receptors that are essential for regulating energy and nutritional homeostasis. PUFAs, being ligands for RXR and PPARs, modulate these and other regulatory events by binding to these nuclear receptor heterodimers. AA, DHA, EPA, LXs, resolvins, and NPD1 have neuroprotective actions [ 4]; inhibit IL-6 and TNF-alpha production that is neurotoxic; and increase synthesis of endothelial nitric oxide (eNO), a neurotransmitter. When neurons are depleted of their PUFA content, as happens after high alcohol intake, formation of LXs, resolvins, and NPD1 will be suboptimal and, hence, neuronal death induced by TNF-alpha cannot be antagonized. This results in fetal alcohol syndrome (FAS). Nicotinamide is necessary for delta-6 desaturase and enhances the formation of various PUFAs and their products [ 8], suggesting that the neuroprotective action of nicotinamide against ethanol-induced FAS, as described by Alessandro Ieraci and Daniel Herrera [ 9], could be attributed to its action on these lipid pathways.

Endocrine-Disrupting Organotin Compounds Are Potent Inducers of Adipogenesis in Vertebrates

Dietary and xenobiotic compounds can disrupt endocrine signaling, particularly of steroid receptors and sexual differentiation. Evidence is also mounting that implicates environmental agents in the growing epidemic of obesity. Despite a long-standing interest in such compounds, their identity has remained elusive. Here we show that the persistent and ubiquitous environmental contaminant, tributyltin chloride (TBT), induces the differentiation of adipocytes in vitro and increases adipose mass in vivo. TBT is a dual, nanomolar affinity ligand for both the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor gamma (PPARgamma). TBT promotes adipogenesis in the murine 3T3-L1 cell model and perturbs key regulators of adipogenesis and lipogenic pathways in vivo. Moreover, in utero exposure to TBT leads to strikingly elevated lipid accumulation in adipose depots, liver, and testis of neonate mice and results in increased epididymal adipose mass in adults. In the amphibian Xenopus laevis, ectopic adipocytes form in and around gonadal tissues after organotin, RXR, or PPARgamma ligand exposure. TBT represents, to our knowledge, the first example of an environmental endocrine disrupter that promotes adipogenesis through RXR and PPARgamma activation. Developmental or chronic lifetime exposure to organotins may therefore act as a chemical stressor for obesity and related disorders.

Efficacy of new retinoids in the prevention of mammary cancers and correlations with short-term biomarkers

A number of retinoid X receptor (RXR) agonists have proven to be highly effective in preventing methylnitrosourea (MNU) induced mammary cancers. However, these agonists have side effects; particularly causing an increase in serum triglyceride levels. A series of ligands for RXR were designed based on computer modeling to the ligand binding domain (LBD) of the RXR receptors and on structure-activity relationships. The chemopreventive effects of these retinoids were evaluated in the relatively long-term MNU model. As a short-term assay to predict their efficacy, the ability of the retinoids to modulate cell proliferation and apoptosis was also determined in mammary cancers after only 7 days of treatment. The five UAB retinoids evaluated included two Class I UAB retinoids (UAB20, UAB112) and three Class II UAB retinoids (UAB30, 4-methyl-UAB30 and the benzosuberone-analog of UAB30). The previously evaluated RXR agonist targretin and the pan-agonist 9-cis-retinoic acid (9-cis-RA), which interacts with both RAR and RXR receptors, were included as positive agonists known to prevent cancer in the MNU model. In the prevention studies, in which the agents were administered beginning 5 days after MNU until the end of the study, targretin (150 mg/kg diet) and 4-methyl-UAB30 (200 mg/kg diet) were highly effective in decreasing cancer numbers by 75-85%. UAB30 (200 mg/kg diet) and 9-cis-RA (60 mg/kg diet) gave intermediate inhibitions of 60 and 45%, respectively. Targretin (15 mg/kg diet), UAB20 (200 mg/kg diet) and the benzosuberone analog of UAB30 (200 mg/kg diet) showed limited activity by decreasing cancer multiplicity 25-30%, while UAB112 had no effect on mammary cancer multiplicity. A direct correlation was observed between the long-term chemopreventive efficacy of these agents and their ability to decrease cell proliferation in mammary cancers after short-term treatment. Furthermore, the highly effective agents (4-methyl-UAB30 and targretin at 150 mg/kg diet) increased apoptosis 3-5 times, while agents with moderate or limited preventive efficacy failed to significantly increase apoptosis. Although the more effective retinoid treatments increased serum triglycerides 2.5- to 4.0-fold, one moderately effective agent (UAB30) had no significant effect on lipid levels. In summary, a short-term in vivo method has been identified for screening newly synthesized retinoids both for chemopreventive efficacy and for their adverse effect on serum triglycerides.

Study of the Combination of Bortezomib with PPAR/RXR Agonists Rosiglitazone and Bexarotene in Lymphoproliferative Disorder-Derived Cell Linesγ.

It has been recently shown that the combination of peroxisome proliferator-activated receptor (PPAR) γ and retinoid X receptor (RXR) ligands enhances their activity as inducers of differentiation and apoptosis. It has also been demonstrated that the induction of the ubiquitin-dependent degradation of PPARγ/RXR heterodimers by the proteosome is activated upon binding of a receptor ligand. Based on this knowledge, we hypothesized that the use of a proteosome inhibitor may enhance the activity of the receptor ligands by prolonging their activation. For this purpose, we tested the antiproliferative activity of the combination of the proteosome inhibitor, bortezomib with PPARγ and RXR ligands, rosiglitazone, and bexarotene as compared to single agents in a series of cell lines derived from ALK+ (JB6, SUPM2)/ALK- (SKB, MAC1) anaplastic large cell lymphoma (ALCL), Burkitt's lymphoma (Hs-Sultan) and multiple myeloma (ARH77). In the combination studies, 5 nM bortezomib was added to cell culture 6 h prior to the addition of 10 μM rosiglitazone and/or 25 μM bexarotene. None of the cell lines reached IC50 as assessed by CellTiter 96 Aqueous cell proliferation assay (Promega), and represented as percent of untreated controls, following 72 h incubation with serial concentrations of rosiglitazone (5, 10, 25, 50 μM) or bexarotene (25 and 50 μM). Bortezomib IC50 was reached only in JB6 cells at 5 nM or in MAC1 and SKB cells at 20 nM at 72 h. The IC50s were reached by 5 nM bortezomib/10 μM rosiglitazone in SUPM2 cells or by 5 nM bortezomib/10 μM rosiglitazone/25 μM bexarotene in Hs-Sultan cells at 72 h. Multiple myeloma-derived cells ARH77 showed resistance to single agent bortezomib, rosiglitazone or bexarotene at all the concentrations tested. However, in these cells, IC50 was reached only by the combination of 5 nM bortezomib/10 μM rosiglitazone/25 μM bexarotene at 96 h of incubation. Surprisingly, ALK+ALCL-derived JB6 cells reached their IC50s with any of the drug combinations (5 nM bortezomib/10 μM rosiglitazone; 5 nM bortezomib/25 μM bexarotene; 5 nM bortezomib/10 μM rosiglitazone/25 μM bexarotene) between 24 (5 nM bortezomib/10 μM rosiglitazone/25 μM bexarotene) and 72 h (5 nM bortezomib/10 μM rosiglitazone; 5 nM bortezomib/25 μM bexarotene). After 72 h of incubation, the percentage of JB6 viable cells treated with 5 nM bortezomib/10 μM rosiglitazone/25 μM bexarotene as compared to untreated controls, was significantly lower than the percentage of cells treated with single agent 5 nM bortezomib (22% ±0.6 vs 45% ± 1.4, P<0.05). In conclusion, our data show that bortezomib at the lowest IC50 (5 nM/JB6 cells) enhances the antiproliferative effects of rosiglitazone or bexarotene in JB6, SUPM2 and Hs-Sultan cells. Ultimately, after 96 h of incubation, the addition of 5 nM bortezomib to the combination of 10 μM rosiglitazone/25 μM bexarotene significantly enhances the antiproliferative activity in myeloma-derived ARH77 cells that showed resistance to single agent bortezomib (39% ±3.8 vs 73% ±1.2, P<0.05).

Selective allosteric ligand activation of the retinoid X receptor heterodimers of NGFI-B and Nurr1

NGFI-B, an orphan member of the NR4A subfamily of the nuclear receptors, recognizes specific sequences in the promoters of neuronal target genes as a monomer. Although NGFI-B also forms a heterodimer with the retinoid X receptor (RXR), a receptor for 9- cis retinoic acid (9CRA), endogenous targets of the heterodimer have not been identified. We investigated the role of RXR ligand binding in NGFI-B/RXR activation and found that dibenzodiazepine-derived ligands, such as the weak RXR agonist HX600, selectively activate NGFI-B/RXR heterodimers. HX600 also activated the heterodimer formed by RXR and Nurr1, another NR4A subfamily receptor. In an assembly assay that detects ligand-dependent reconstruction of the ligand-binding domain, HX600 and not 9CRA induced an allosteric ligand effect on NGFI-B through RXRα binding. The data indicate that the RXR heterodimers of NGFI-B and Nurr1 are selectively activated by the RXR ligand HX600, and that compounds such as HX600 will be valuable tools in investigating NGFI-B and Nurr1 function.

Prostate cancer cell type-specific involvement of the VDR and RXR in regulation of the human PTHrP gene via a negative VDRE

Parathyroid hormone-related protein (PTHrP) increases the growth and osteolytic potential of prostate cancer cells, making it important to control PTHrP expression in these cells. We show that 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) and its non-hypercalcemic analog, EB1089, decrease PTHrP mRNA and cellular protein levels in the androgen-dependent human prostate cancer cell line LNCaP and its androgen-independent derivative, the C4-2 cell line. This effect is mediated via a negative Vitamin D response element (nVDRE hPTHrP) within the human PTHrP gene and involves an interaction between nVDRE hPTHrP and the Vitamin D receptor (VDR). The retinoid X receptor (RXR) is a frequent heterodimeric partner of the VDR. We show that RXRα forms part of the nuclear protein complex that interacts with nVDRE hPTHrP along with the VDR in LNCaP and C4-2 cells. We also show that the RXR ligand, 9- cis-retinoic acid, downregulates PTHrP mRNA levels; this decrease is more pronounced in LNCaP than in C4-2 cells. In addition, 9- cis-retinoic acid enhances the 1,25(OH) 2D 3-mediated downregulation of PTHrP expression in both cell lines; this effect also is more pronounced in LNCaP cells. Proliferation of LNCaP, but not C4-2, cells is decreased by 9- cis-retinoic acid. Promoter activity driven by nVDRE hPTHrP cloned upstream of the SV40 promoter and transiently transfected into LNCaP and C4-2 cells is downregulated in response to 1,25(OH) 2D 3 and EB1089 in both cell lines. Co-treatment with these compounds and 9- cis-retinoic acid further decreases CAT activity in LNCaP, but not C4-2, cells. These results indicate that PTHrP gene expression is regulated by 1,25(OH) 2D 3 in a cell type-specific manner in prostate cancer cells.

Crystal Structure of a Novel Tetrameric Complex of Agonist-bound Ligand-binding Domain of Biomphalaria glabrata Retinoid X Receptor

Nuclear receptors form an important class of transcription regulators in metazoans. To learn more about the evolution of these proteins, we have initiated structural studies on nuclear receptor ligand-binding domains from various animals. Here we present the crystal structure of the ligand-binding domain (LBD) of the retinoid X receptor (RXR) from the mollusc Biomphalaria glabrata. The structure reveals a novel tetrameric association in which each monomer is complexed to the human RXR ligand 9-cis retinoic acid and to a human co-activator-derived peptide. The ligand and the co-activator peptide are bound in essentially the same manner as observed in previously reported human RXR LBD structures, suggesting that the mechanisms of RXR-mediated transcription regulation are very similar in mollusc and human. The structure shows further that binding of ligand and co-activator peptide does not necessarily lead to the typical holo-conformation in which helix 12 (H12) folds back and packs against the LBD. Within a canonical dimer, only one monomer is in this closed agonist conformation. The other monomer is in an open conformation with H12 protruding from the LBD core, occupying the H12 interaction groove of another open monomer in an adjacent dimer in a domain swapping fashion, thus resulting in a tetrameric association. Additional tetramer interfaces are formed between H11 of the closed LBD and H6 of the open LBD. This novel holo-tetramer configuration may have a biological role in activating genes whose promoters are poorly recognised by dimers but much more efficiently by the corresponding tetramers.

The human ABCG1 gene: identification of LXR response elements that modulate expression in macrophages and liver

The ABC transporter ABCG1 (ATP binding cassette transporter G1), expressed in macrophages, liver, and other tissues, has been implicated in the efflux of cholesterol to high density lipoprotein. The ABCG1 gene is transcriptionally activated by cholesterol loading and activators of liver X receptors (LXRs) and retinoid X receptors (RXRs) through genomic sequences that have not been fully characterized. Here we show that ABCG1 mRNA is induced by LXR agonists in RAW264.7 macrophage cells, HepG2 hepatoma cells, and primary mouse hepatocytes. We identify two evolutionarily highly conserved LXR response elements (LXREs), LXRE-A and LXRE-B, located in the first and second introns of the human ABCG1 gene. Each element conferred robust LXR-agonist responsiveness to ABCG1 promoter-directed luciferase gene constructs in RAW264.7 and HepG2 cells. Overexpression of LXR/RXR activated the ABCG1 promoter in the presence of LXRE-A or LXRE-B sequences. In gel-shift assays, LXR/RXR heterodimers bound to wild-type but not to mutated LXRE-A and LXRE-B sequences. In chromatin immunoprecipitation assays, LXR and RXR were detected at LXRE-A and -B regions of DNA of human THP-1 macrophages. These studies clarify the mechanism of transcriptional upregulation of the ABCG1 gene by oxysterols in macrophages and liver, two key tissues where ABCG1 expression may affect cholesterol balance and atherogenesis.

HX531, a retinoid X receptor antagonist, inhibited the 9-cis retinoic acid-induced binding with steroid receptor coactivator-1 as detected by surface plasmon resonance

HX531 is a retinoid X receptor (RXR) antagonist that inhibits 9-cis retinoic acid-induced neutrophilic differentiation of HL-60 cells. In order to elucidate the inhibitory mechanism of HX531, we have developed a novel ligand sensor assay for RXR in which the receptor–coactivator interaction is directly monitored using surface plasmon resonance (SPR) biosensor technology. A 20-mer peptide from steroid receptor coactivator-1 (SRC-1), containing nuclear receptor interaction motif LXXLL was immobilized on the surface of a BIAcore sensor chip. Injection of human recombinant RXR with or without 9-cis retinoic acid resulted in ligand-dependent interaction with the SRC-1 peptide. Kinetic analysis revealed dissociation constants (KD) of 9-cis RA-preincubated RXR to SRC-1 was 5.92 × 10 −8 M. Using this technique, we found that 1 μM HX531 reduced the ka value of liganded-RXR with SRC-1, suggesting that HX531 reduced the affinity of RXR to SRC-1. This SPR assay system was applied to obtain quantitative kinetic data of RXR ligand binding to the SRC-1 peptide and the alteration of these data by antagonists.

Expression and regulation of sterol 27-hydroxylase (CYP27A1) in human macrophages: a role for RXR and PPARγ ligands

CYP27A1 (sterol 27-hydroxylase) catalyses an important sterol elimination pathway in the human macrophage, and consequently may protect against atherosclerosis. We studied the expression and regulation of CYP27A1 in a human macrophage-like cell-line, THP-1, and primary HMDMs (human monocyte-derived macrophages). In both macrophage cell types, we found that CYP27A1 expression is independent of cellular cholesterol levels and of LXR (liver X receptor)-dependent control of transcription. However, the RXR (retinoid X receptor) ligand, 9-cis-retinoic acid, upregulates CYP27A1 expression. Of the RXR heterodimeric partners tested, PPAR (peroxisome-proliferator-activated receptor) gamma ligands significantly increased CYP27A1 mRNA levels. Its reversal by a PPARgamma antagonist demonstrated the specificity of this effect. Interestingly, HMDMs express markedly higher levels of CYP27A1 than THP-1 macrophages, and this difference was reflected in both protein levels and enzyme activities between the two cell types. In conclusion, stimulation of CYP27A1 by PPARgamma may represent a key previously unrecognized mechanism by which PPARgamma protects against atherosclerosis.

The Nuclear Xenobiotic Receptor CAR: Structural Determinants of Constitutive Activation and Heterodimerization

Constitutive androstane receptor (CAR) induces xenobiotic, bilirubin, and thyroid hormone metabolism as a heterodimer with the retinoid X receptor (RXR). Unlike ligand-dependent nuclear receptors, CAR is constitutively active. Here, we report the heterodimeric structure of the CAR and RXR ligand binding domains (LBDs), which reveals an unusually large dimerization interface and a small CAR ligand binding pocket. Constitutive CAR activity appears to be mediated by the compact nature of the CAR LBD that displays several unique features including a shortened AF2 helix and helix H10, which are linked by a two-turn helix that normally adopts an extended loop in other receptors, and an extended helix H2 that stabilizes the canonical LBD fold by packing tightly against helix H3. These structural observations provide a molecular framework for understanding the atypical transcriptional activation properties of CAR.

Molecular interaction of retinoic acid receptors with coregulators PCAF and RIP140

p300/CBP-associating factor (PCAF) is a ligand-dependent coactivator, whereas receptor-interacting protein 140 (RIP140) is a ligand-dependent negative coregulator for retinoic acid (RA) receptor (RAR) and retinoid X receptor (RXR). To compare these molecular interactions and to determine the effect of RXR ligands, we focus on PCAF/RAR/RXR complex formation in this study for a comparison to RIP140/RAR/RXR complex formation. The LBD of RXR is identified as its primary PCAF-interacting motif. BIAcore studies determine the K d of RAR/RXR association with PCAF as 9.35 nM in the presence of RXR ligand AGN194204, and 47.2 nM in the absence of ligand. Cross-linking study demonstrates tri-molecular complex consisting of one RAR/RXR pair and one PCAF. In competition experiments, RIP140 strongly competes with PCAF for interaction with RAR/RXR both in vitro and in vivo. Chromatin immunoprecipitation demonstrates recruitment of RIP140 and PCAF to the endogenous RA-regulated gene, the RARβ2 promoter. This study presents kinetic evidence for competition of RIP140 with PCAF for ligand-dependent interactions with RAR/RXR, and provides kinetic explanation for the suppressive activity of RIP140 in RA-activated gene expression.

Nurr1, an orphan nuclear receptor with essential functions in developing dopamine cells.

Nurr1 is a transcription factor that is expressed in the embryonic ventral midbrain and is critical for the development of dopamine (DA) neurons. It belongs to the conserved family of nuclear receptors but lacks an identified ligand and is therefore referred to as an orphan receptor. Recent structural studies have indicated that Nurr1 belongs to a class of ligand-independent nuclear receptors that are unable to bind cognate ligands. However, Nurr1 can promote signaling via its heterodimerization partner, the retinoid X receptor (RXR). RXR ligands can promote the survival of DA neurons via a process that depends on Nurr1-RXR heterodimers. In developing DA cells, Nurr1 is required for the expression of several genes important for DA synthesis and function. However, Nurr1 is probably also important for the maintenance of adult DA neurons and plays additional less-well-elucidated roles in other regions of the central nervous system and in peripheral tissues.

A retinoid X receptor (RXR)-selective retinoid reveals that RXR-alpha is potentially a therapeutic target in breast cancer cell lines, and that it potentiates antiproliferative and apoptotic responses to peroxisome proliferator-activated receptor ligands.

IntroductionCertain lipids have been shown to be ligands for a subgroup of the nuclear hormone receptor superfamily known as the peroxisome proliferator-activated receptors (PPARs). Ligands for these transcription factors have been used in experimental cancer therapies. PPARs heterodimerize and bind DNA with retinoid X receptors (RXRs), which have homology to other members of the nuclear receptor superfamily. Retinoids have been found to be effective in treating many types of cancer. However, many breast cancers become resistant to the chemotherapeutic effects of these drugs. Recently, RXR-selective ligands were discovered that inhibited proliferation of all-trans retinoic acid resistant breast cancer cells in vitro and caused regression of the disease in animal models. There are few published studies on the efficacy of combined therapy using PPAR and RXR ligands for breast cancer prevention or treatment.MethodsWe determined the effects of selective PPAR and RXR ligands on established human breast cancer cell lines in vitro.ResultsPPAR-α and PPAR-γ ligands induced apoptotic and antiproliferative responses in human breast cancer cell lines, respectively, which were associated with specific changes in gene expression. These responses were potentiated by the RXR-selective ligand AGN194204. Interestingly, RXR-α-overexpressing retinoic acid resistant breast cancer cell lines were more sensitive to the effects of the RXR-selective compound.ConclusionRXR-selective retinoids can potentiate the antiproliferative and apoptotic responses of breast cancer cell lines to PPAR ligands.