Receptor RXR Research Articles

A randomized phase III trial comparing bexarotene/cisplatin/vinorelbine versus cisplatin/vinorelbine in chemotherapy-naive patients with advanced or metastatic non-small cell lung cancer (NSCLC)

LBA7024 Background: Targretin (bexarotene) is a rexinoid that selectively activates RXR receptors which modulate functions associated with differentiation, inhibition of cell growth, apoptosis and metastasis. Data from four Phase I and Phase II studies suggests that combining bexarotene with chemotherapy in patients with first-line NSCLC may prolong survival. A randomized Phase III trial was performed to determine if adding bexarotene to cisplatin/vinorelbine chemotherapy prolongs survival of NSCLC patients. Methods: 623 chemo-naive NSCLC patients who had Stage IIIb disease with pleural effusion or Stage IV disease, who were performance status ECOG 0–1, and who had normalized triglyceride levels at study entry were enrolled. This study size will allow detection of a 30% survival improvement in the bexarotene treatment arm. Patients were stratified by disease stage and gender prior to randomization to bexarotene (400mg/m2/day) with cisplatin/vinorelbine or cisplatin/vinorelbine alone. Overall survival distribution (primary endpoint) between the arms will be analyzed by stratified log-rank test. Secondary endpoint is the one-year survival rate. Safety assessments were conducted. Efficacy and safety data will be mature and available in March, 2005. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Ligand Ligand Ligand Ligand

5 The role of nuclear retinoid receptors RAR and RXR in human eosinophil functions

5 The role of nuclear retinoid receptors RAR and RXR in human eosinophil functions

Human ADA3 Binds to Estrogen Receptor (ER) and Functions As a Coactivator for ER-mediated Transactivation

We have recently identified the hADA3 protein, the human homologue of yeast transcriptional coactivator yADA3, as a novel HPV16 E6 target. Using ectopic expression approaches, we further demonstrated that hADA3 directly binds to the 9-cis retinoic acid receptors alpha and beta, and functions as a coactivator for retinoid receptor-mediated transcriptional activation. Here, we examined the role of endogenous hADA3 as a coactivator for estrogen receptor (ER), an important member of the nuclear hormone receptor superfamily. We show that ADA3 directly interacts with ER alpha and ER beta. Using the chromatin immunoprecipitation assay, we also show that hADA3 is a component of the activator complexes bound to the native ER response element within the promoter of the estrogen-responsive gene pS2. Furthermore, using an ER response element-luciferase reporter, we show that overexpression of ADA3 enhances the ER alpha- and ER beta-mediated sequence-specific transactivation. Reverse transcription-PCR analysis showed an ADA3-mediated increase in estrogen-induced expression of the endogenous pS2 gene. More importantly, using RNA interference against hADA3, we demonstrate that inhibition of endogenous hADA3 inhibited ER-mediated transactivation and the estrogen-induced increase in the expression of pS2, cathepsin D, and progesterone receptor, three widely known ER-responsive genes. The HPV E6 protein, by targeting hADA3 for degradation, inhibited the ER alpha-mediated transactivation and the protein expression of ER target genes. Thus, our results demonstrate that ADA3 directly binds to human estrogen receptor and enhances the transcription of ER-responsive genes, suggesting a broader role of mammalian hADA3 as a coactivator of nuclear hormone receptors and the potential role of these pathways in HPV oncogenesis.

Efficacy of Targretin on methylnitrosourea-induced mammary cancers: prevention and therapy dose-response curves and effects on proliferation and apoptosis

Various aspects of the chemopreventive and chemotherapeutic properties of the RXR receptor agonist Targretin (LGD 1069) were examined in the methylnitrosourea (MNU)-induced model of mammary cancer. The administration of Targretin at dose levels of 60, 20 or 6.7 mg/kg body wt/day by gavage decreased the number of mammary tumors by 96, 85 and 78%, respectively. When Targretin was administered in the diet at 92 and 275 mg/kg diet cancer multiplicities were reduced by 78 and 92%, respectively. A wider range of dietary doses of Targretin at 15, 50 and 150 mg/kg diet reduced the number of mammary tumors by 38, 55 and 70%, respectively. Treatment of rats with different regimens of Targretin (250 mg/kg diet) yielded cancer multiplicities of 4.3 for non-treated rats, 0.5 for rats treated continuously with Targretin, 2.1 for rats treated with Targretin for 8 weeks followed by 10 weeks of the control diet and 1.6 for rats treated with Targretin alternating 3 days on and 4 days off. Targretin was also examined as a therapeutic agent by treating rats with at least one palpable mammary tumor for 5 weeks. A high dose of Targretin (272 mg/kg diet) caused partial or complete regression of approximately 65% of the cancers over this time period. In contrast, in animals treated with 15 mg Targretin/kg diet only 1 of 12 cancers showed significant regression. Finally, the effect of a limited exposure to Targretin (7 days) on cell proliferation and apoptosis in small mammary tumors was determined. Targretin at 150 mg/kg diet strongly decreased proliferation (75%) and increased apoptosis (300%), while a lower dose of Targretin (15 mg/kg diet, which still prevented 30% of cancers) had no effect on apoptosis but did decrease cell proliferation. Determination of serum IGF1 levels showed that treatment of rats with highly effective doses of Targretin at 272 mg/kg diet or at 60 or 20 mg/kg body wt/day by gavage caused significantly decreased serum IGF1 levels.

The novel synthetic oleanane triterpenoid CDDO (2-cyano-3, 12-dioxoolean-1, 9-dien-28-oic acid) induces apoptosis in Mycosis fungoides/Sézary syndrome cells.

The novel synthetic oleanane triterpenoid CDDO (2-cyano-3, 12-dioxoolean-1, 9-dien-28-oic acid) can serve as a ligand for the peroxisome proliferator activator receptor-gamma (PPAR-gamma) and has been shown to inhibit cell proliferation, and to induce differentiation and apoptosis in tumor cell lines. Bexarotene is an RXR-selective retinoid that can induce apoptosis of mycosis fungoides (MF) and Sézary syndrome (SS) cells. Since the PPAR-gamma and RXR receptors can form heterodimers, we studied the effects of CDDO and its synergism with bexarotene on apoptosis in MF/SS cell lines (MJ, Hut78, and HH) and freshly isolated peripheral blood lymphocytes (PBL) from SS patients with circulating atypical T cells (CD4+CD26-). CDDO treatment at 1-5 microM for 48 h caused a concentration-dependent apoptosis in three MF/SS cell lines and patients' PBL compared to vehicle controls. Bexarotene augmented CDDO-induced apoptosis in these cells. PPAR-gamma was expressed but decreased by 47% in MJ, 42% in Hut78, and 77% in HH cells following CDDO treatment. The anti-apoptotic protein bcl-xL, but not bcl-2, was decreased by 69% in MJ, 31% in Hut78, and 59% in HH cells and caspase-3 was activated following CDDO treatment. Interestingly, the PPAR-gamma antagonist T007 did not block CDDO-induced apoptosis, and the more potent PPAR-gamma agonist rosiglitazone required much higher concentrations (>50 microM) than CDDO to induce apoptosis in MF/SS cells. In summary, CDDO induces apoptosis that is further enhanced by bexarotene and decreases the PPAR-gamma and bcl-xL proteins in MF/SS cells. CDDO's effects on MF/SS cells may be at least partly mediated through a PPAR-gamma-independent mechanism. Our findings suggest the rationale for further investigation of the clinical potential of CDDO, either alone or in combination with bexarotene for MF/SS patients.

Potentiation of the teratogenic effects induced by coadministration of retinoic acid or phytanic acid/phytol with synthetic retinoid receptor ligands

Previous studies in our laboratory identified retinoid-induced defects that are mediated by RAR-RXR heterodimerization using interaction of synthetic ligands selective for the retinoid receptors RAR and RXR in mice (Elmazar et al. 1997, Toxicol Appl Pharmacol 146:21-28; Elmazar et al. 2001, Toxicol Appl Pharmacol 170:2-9; Nau and Elmazar 1999, Handbook of experimental pharmacology, vol 139, Retinoids, Springer-Verlag, pp 465-487). The present study was designed to investigate whether these RAR-RXR heterodimer-mediated defects can be also induced by interactions of natural and synthetic ligands for retinoid receptors. A non-teratogenic dose of the natural RXR agonist phytanic acid (100 mg/kg orally) or its precursor phytol (500 mg/kg orally) was coadministered with a synthetic RARalpha-agonist (Am580; 5 mg/kg orally) to NMRI mice on day 8.25 of gestation (GD8.25). Furthermore, a non-teratogenic dose of the synthetic RXR agonist LGD1069 (20 mg/kg orally) was also coadministered with the natural RAR agonist, all- trans-retinoic acid (atRA, 20 mg/kg orally) or its precursor retinol (ROH, 50 mg/kg orally) to NMRI mice on GD8.25. The teratogenic outcome was scored in day-18 fetuses. The incidence of Am580-induced resorptions, spina bifida aperta, micrognathia, anotia, kidney hypoplasia, dilated bladder, undescended testis, atresia ani, short and absent tail, fused ribs and fetal weight retardation were potentiated by coadministration of phytanic acid or its precursor phytol. Am580-induced exencephaly and cleft palate, which were not potentiated by coadministration with the synthetic RXR agonists, were also not potentiated by coadministration with either phytanic acid or its precursor phytol. LGD1069 potentiated atRA- and ROH-induced resorption, exencephaly, spina bifida, aperta, ear anotia and microtia, macroglossia, kidney hypoplasia, undescended testis, atresia ani, tail defects and fetal weight retardation, but not cleft palate. These results suggest that synergistic teratogenesis can be induced by coadministration of a natural RXR ligand (phytanic acid) with a synthetic RAR agonist (Am580). Thus, certain potentially useful therapeutic agents or nutritional factors such as phytanic acid should be tested for teratogenic risk by coadministration with other retinoid receptor agonists.

Improved event free survival (EFS) with the novel retinoid, bexarotene (BEX) and gemcitabine/carboplatin (G/C) in non-small cell lung cancer (NSCLC)

7104 Background: BEX (Targretin) is a novel retinoid targeting the RXR receptor. RXR expression has been correlated with survival in NSCLC. Phase I/II data have indicated prolonged survival in NSCL...

Phase I/II study of bexarotene in combination with weekly paclitaxel and monthly carboplatin for the treatment of patients (pts) with advanced non-small cell lung cancer (NSCLC)

7356 Background: This trial was based on a multicenter randomized phase II trial in advanced NSCLC where weekly paclitaxel and monthly carboplatin demonstrated favorable results. This trial was designed to combine this chemotherapy regimen with bexarotene, a retinoid that binds selectively to the nuclear receptor RXR now in phase III development. Methods: Pt eligibility: pathologic stage: IIIB/IV or recurrent metastatic disease, adequate hematological, hepatic, thyroid, lipid and renal function. Prior chemotherapy was allowed. Bexarotene oral capsules are administered daily starting on initial day of chemotherapy. Two dose levels of bexarotene were evaluated (300 mg/m2/day and 400 mg/m2/day). All patients were scheduled to receive paclitaxel 100 mg/m2 weekly for 3 doses every 4 weeks and carboplatin AUC = 6 monthly. Results: As of December 2003, 21 pts were enrolled, 8 receiving 300mg/m2/day and 13 at 400 mg/m2/day of bexarotene. Pt characteristics include: age (median 58), gender (female 43%), stage (86% stage IV), 24% Karnofsky performance status 60–70%, 29% prior chemotherapy, 33% prior radiation, 48% prior surgery. At the 300 mg/m2/day bexarotene dose level, 52% of the pts required a dose reduction in paclitaxel . At the 400 mg/m2/day dose level of bexarotene, 34% of the pts acquired a dose reduction in paclitaxel. Dose modification rates were higher in our study (52%/34%) than in the prior randomized study without bexarotene (14%). The primary serious adverse events (grade 3 or 4) were neutropenia (19%), hypertriglyceridemia (14%) and anemia (10%). Conclusions: Daily administration of bexarotene at 400 mg/m2/day in combination with weekly paclitaxel and monthly carboplatin is safe and feasible. Enrollment of chemotherapy naïve pts into the phase II portion of this trial continues. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Ligand Ligand Ligand; Genentech; Abbott; Amgen; Aventis; Bristol-Myers Squibb; GlaxoSmithKline; Ortho Biotech; Merck; Pfizer; OSI; Roche

Phase I/II study of bexarotene in combination with weekly paclitaxel and monthly carboplatin for the treatment of patients (pts) with advanced non-small cell lung cancer (NSCLC)

7356 Background: This trial was based on a multicenter randomized phase II trial in advanced NSCLC where weekly paclitaxel and monthly carboplatin demonstrated favorable results. This trial was designed to combine this chemotherapy regimen with bexarotene, a retinoid that binds selectively to the nuclear receptor RXR now in phase III development. Methods: Pt eligibility: pathologic stage: IIIB/IV or recurrent metastatic disease, adequate hematological, hepatic, thyroid, lipid and renal function. Prior chemotherapy was allowed. Bexarotene oral capsules are administered daily starting on initial day of chemotherapy. Two dose levels of bexarotene were evaluated (300 mg/m2/day and 400 mg/m2/day). All patients were scheduled to receive paclitaxel 100 mg/m2 weekly for 3 doses every 4 weeks and carboplatin AUC = 6 monthly. Results: As of December 2003, 21 pts were enrolled, 8 receiving 300mg/m2/day and 13 at 400 mg/m2/day of bexarotene. Pt characteristics include: age (median 58), gender (female 43%), stage (86% stage IV), 24% Karnofsky performance status 60–70%, 29% prior chemotherapy, 33% prior radiation, 48% prior surgery. At the 300 mg/m2/day bexarotene dose level, 52% of the pts required a dose reduction in paclitaxel . At the 400 mg/m2/day dose level of bexarotene, 34% of the pts acquired a dose reduction in paclitaxel. Dose modification rates were higher in our study (52%/34%) than in the prior randomized study without bexarotene (14%). The primary serious adverse events (grade 3 or 4) were neutropenia (19%), hypertriglyceridemia (14%) and anemia (10%). Conclusions: Daily administration of bexarotene at 400 mg/m2/day in combination with weekly paclitaxel and monthly carboplatin is safe and feasible. Enrollment of chemotherapy naïve pts into the phase II portion of this trial continues. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Ligand Ligand Ligand; Genentech; Abbott; Amgen; Aventis; Bristol-Myers Squibb; GlaxoSmithKline; Ortho Biotech; Merck; Pfizer; OSI; Roche

813. Ecdysone Receptor-Based Inducible Gene Regulation System for Simultaneous Regulation of Multiple Genes

An ecdysone receptor (EcR)-based inducible gene regulation system that functions in mammalian cells in a tightly regulated fashion is described. This gene switch functions efficiently in a two-hybrid format in which heterologous DNA binding and transcription activation domains are fused to the two heterodimeric receptor partners EcR and RXR. Extensive mutagenesis of the ecdysone receptor along with the development and screening of a large number of non-steroid ligands has resulted in the discovery of several orthogonal ligand-receptor pairs. Receptor mutants were obtained that are activated at low nanomolar concentrations of the ligand. The different components of the gene switch were assembled into a two-vector format or into a single cassette all-in-one vector for expression of genes of interest. Different ecdysone receptor mutants were fused to different DNA binding domains to create several independently acting gene switch combinations (multiplex). The orthogonal nature of the ligand-receptor pairs was demonstrated in reporter assays of two-channel gene switch combinations. Experiments will be presented to demonstrate that the multiplex gene switch system works independently in single cells in response to different ligands. The utility of this system in gene therapy applications will be discussed.

Ligand specificity and developmental expression of RXR and ecdysone receptor in the migratory locust

The ecdysone receptor 1 1 The avoid ambiguity in the terminology relating to this system, we have followed the usage suggested by Riddiford et al. (2001) under which the heterodimer is designated the ecdysone receptor and one of its components is referred to as EcR. , which is a heterodimer of EcR and the retinoic acid receptor (RXR) homolog, Ultraspiracle (USP), has been well studied in the evolutionarily advanced and derived insects, the flies and moths. It is less well characterized in more primitive insect orders such as the Orthoptera, which include the grasshoppers and locusts. Following our previous isolation from Locusta migratoria (Lm) of a shorter RXR isoform (now called LmRXR-S), the isolation of a second, longer isoform (LmRXR-L) that appears to have characteristics of a ligand-modulated nuclear receptor is reported here. Transcripts for both isoforms, as well as LmEcR, were detected in embryos and in females during oocyte maturation. After expression in E. coli, both LmRXR-S and LmRXR-L form heterodimers with recombinant LmEcR in vitro which bind the active ecdysteroid, ponasterone A. Binding was only weakly competed for by ecdysone agonists that are known to be toxic to more advanced insects, suggesting functionally significant divergence in EcR ligand binding domains. In contrast, the DNA binding domain of LmEcR is less divergent and a protein complex, presumably LmEcR/LmRXR, that bound the ecdysone response element, IR-1, was detected in locust nuclear extracts. Because of reports of juvenile hormone (JH III) binding to Drosophila USP and the observed in silico RXR-like ligand-binding site in LmRXR-L, the recombinant proteins were also tested for binding to JH III. Neither LmRXR isoform, alone or in combination with LmEcR, bound JH III at nanomolar concentrations.

Nuclear substructure and dynamics

Many aspects of nuclear organization exhibit dynamic properties. Both nuclear bodies and individual chromatin regions can move through the nucleoplasm. Certain nuclear bodies are able to assemble and disassemble from soluble components in response to the metabolic demands of the cell. The cycling behaviour of nuclear factors and the rapid turnover of nuclear body components also underlines the importance of measuring the dynamic properties of proteins and RNPs when interpreting both localization patterns and their possible functional roles. Static fluorescence or electron microscopy images usually indicate steady state accumulations of nuclear factors, but can fail to reveal sites where factors localize transiently. Recent work has also shown that some nuclear proteins and RNPs participate in directional trafficking pathways, moving in a temporal sequence between distinct classes of nuclear bodies. For example, the nucleolar snoRNP protein NHPX/p15.5kDa transiently accumulates in splicing speckles before adopting a steady state concentration in nucleoli and Cajal bodies.Our understanding of the dynamic organization of the cell nucleus is expanding rapidly and this is facilitated by the increasing range of new methods to study the composition, structure and movement of nuclear bodies and their components. In the case of nucleoli, NPCs, speckles and Cajal bodies, they can be purified and their protein components can be identified using mass spectrometry. The ability to label nuclear structures in vivo through the expression of fluorescent protein-tagged factors also now allows many dynamic parameters to be measured directly in living cells using photobleaching techniques. Further developments in quantitative fluorescence microscopy and quantitative proteomics will help to develop a more robust model of how the nucleus is organized and should inform our view as to how nuclear structure influences cellular function.

From CHD Susceptibility Genes to New Therapeutics

The identification of genetic disease susceptibility genes has been pursued with the expectation to rapidly translate causal disease mechanisms into innovative therapeutics. Indeed, several susceptibility genes have led to the elucidation of molecular pathologies and the development of new drugs and diagnostics for disease management. We review in this article how the identification of susceptibility genes for high density lipoprotein (HDL) particle deficiencies have changed our understanding of lipid metabolism and how this understanding is being used in translational research for the discovery of new treatment strategies. The identification of ATP-binding cassette transporter A1 (ABCA1) and transcriptional repressor zinc finger protein 202 (ZNF202) as susceptibility genes for coronary heart disease (CHD) and hypoalphalipoproteinemia, led to the characterization of a functional network of genes involved in lipid metabolism. Currently available drug classes for metabolic and dyslipidemic indications such as the glitazones, fibrates and statins have been shown to modulate the expression of some components involved in reverse cholesterol transport. The characterization of new regulators of this gene network that are amenable to pharmaceutical modulation such as the nuclear receptors FXR, LXR, PXR, PPAR and RXR may offer new perspectives in disease treatment, if undesirable side effects can be diminished. In addition, the characterization of ABCA1-mediated lipid efflux systems has provided new biomarkers to assess the pharmacological effects of new therapeutic strategies on reverse cholesterol transport. Keywords: chd susceptibility genes, atp-binding cassette transporter a1, transcriptional repressor zinc finger, coronary heart disease, hypoalphalipoproteinemia

Correlation between Retinoic Acid Sensitivity and IGFBP-3, AFP Protein Expression in Hepatoma Cell Lines.

Retinoic acid (RA) has been known to inhibit the proliferation, and to induce apoptosis, of various cancer cell lines. We investigated the correlation between the protein levels of the RAR and RXR receptor families, IGFBP-3 and AFP, and the RA sensitivity in hepatoma cell lines. The cell growth inhibition was examined by assaying various 1 to 10muM RA treated hepatoma cell lines. Western blot analysis for the RAR and RXR families, AFP and IGFBP-3 were performed after treatment with 10muM RA. The 1 to 10muM RA treatment induced growth inhibition in the SNU368, SNU354, SNU398 and HepG2 cells. The cell growth of SNU449 and Hep3B were not suppressed by 1muM, but were slightly suppressed by 10muM RA. An increased expression of IGFBP-3 in HepG2, SNU354, SNU398 and SNU368 cells, and a decreased expression of alpha-fetoprotein (AFP), was observed from the western blot analysis in all hepatoma cells tested, whereas no confirmed tendency of RAR expressions was seen. Our result showed that the growth inhibition of RA differed according to the sensitivity of the type of cells to RA. We supposed that RA-induced cell growth inhibition may be related to the expressions of IGFBP-3 and AFP, but no exact correlation exists between the growth inhibition and receptor expression status in hepatoma cell lines.

Design and Synthesis of Receptor Ligands

Publisher Summary This chapter focuses on the design and synthesis of receptor ligands. The synthetic ligands of nuclear receptors have a number of properties that make them useful as pharmacological probes in the study of nuclear receptor action. These properties include selective action, either in specifically binding to a receptor orthologue, isoform, or subtype; the selective activation of specific hormone response elements; or antagonism, where a ligand competes with the natural hormone for binding to the receptor but does not activate it. Nuclear hormone receptors play important roles in development and homeostasis and are implicated in a variety of diseases such as cancer, diabetes, and various endocrine disorders. The roles of the RXR, TR, LXR, FXR, and PPAR receptors in regulating lipid homeostasis also make them potentially attractive targets to treat or prevent cardiovascular diseases. The selective activators (or inhibitors) of nuclear receptors have the promise to reduce these side effects, thereby acting as safer and more useful drugs. The classic steroid hormones are derived from cholesterol by enzymatic modification. Other cholesterol metabolites, including oxysterols and bile acids, serve as ligands for LXR and FXR, respectively. Retinoids are produced from the oxidative cleavage of β-carotene, an essential nutrient. While natural ligands of NRs are produced by a variety of different pathways, the ligands themselves share common features. They are largely hydrophobic in character, consisting mostly of aliphatic, aromatic, or olefinic hydrocarbons.

Prevention of vitamin A teratogenesis by phytol or phytanic acid results from reduced metabolism of retinol to the teratogenic metabolite, all-trans-retinoic acid.

Previous studies in our laboratory showed a synergistic interaction of synthetic ligands selective for the retinoid receptors RAR and RXR in regard to teratogenic effects produced in mice (M. M. Elmazar et al., 2001, TOXICOL: Appl. Pharmacol. 170, 2-9). In the present study the influence of phytol and phytanic acid (a RXR-selective ligand) on the teratogenicity of retinol and the RAR-selective ligand all-trans-retinoic acid was investigated by coadministration experiments on day 8.25 of gestation in NMRI mice. Phytol and phytanic acid, noneffective when administered alone, did not potentiate the teratogenicity induced by retinol or all-trans-retinoic acid. On the contrary, phytol and phytanic acid greatly reduced retinol-induced teratogenic effects (ear anotia, tail defects, exencephaly). The effect of phytol on all-trans-retinoic acid teratogenesis was limited (only resorptions and tail defects were reduced). Pharmacokinetic studies in nonpregnant animals revealed that phytol coadministration with retinol reduced plasma levels of retinol and retinyl esters, and drastically reduced the levels of the teratogenic retinol metabolite, all-trans-retinoic acid. Phytanic acid also reduced the oxidative metabolism and teratogenic effects of retinol. These results indicate that phytol and phytanic acid did not synergize with retinol and all-trans-retinoic acid in our mouse teratogenesis model. Instead, phytol and phytanic acid effectively blocked the teratogenic effects of retinol by drastically reducing the metabolic production of all-trans-retinoic acid. Phytol and phytanic acid may be useful for the prevention of vitamin A teratogenicity.

The Human ABCG4 Gene Is Regulated by Oxysterols and Retinoids in Monocyte-Derived Macrophages

Here we report the induction of gene expression of ABCG4, a member of the ABC transporter subfamily G, from human macrophages by oxysterols and retinoids, agonists of the nuclear receptors LXR and RXR. The cloned ABCG4 transcript has a size of 3.5 kb and contains an open reading frame which encodes a polypeptide of 646 amino acids. Structurally, the putative ABC transporter protein consists of a nucleotide binding fold followed by a cluster of six transmembrane-spanning domains and thus conforms to the group of half-size ABC transporters. Among the human ABC transporter subfamily G members the novel transporter shows highest protein sequence homology and identity to ABCG1 (84 and 72%, respectively). Analysis of the genomic organization demonstrates that the ABCG4 gene is composed of at least 14 exons which extend across a region of 12.6 kb in size on chromosome 11q23.3. Based on its structural features and an LXR/RXR-responsive regulation similar to the cellular lipid export protein ABCA1, we conclude that ABCG4 may be involved in macrophage lipid homeostasis.

Retinoic acid causes MEK-dependent RAF phosphorylation through RARα plus RXR activation in HL-60 cells

Retinoic acid (RA) is known to cause the myeloid differentiation of HL-60 human myeloblastic leukemia cells in a process requiring MEK-dependent ERK2 activation. This RA-induced ERK2 activation appears after approximately 4 h and persists until the cells are differentiated and G0 arrested (Yen et al, 1998). This motivates the question of whether RA also activated RAF as part of a typical RAF/MEK/MAPK cascade. Retinoic acid is shown here to also increase the phosphorylation of RAF, but in an unusual way. Surprisingly, increased RAF phosphorylation is first detectable after 12 to 24 hours by phosphorylation-induced retardation of polyacrylamide gel electrophoretic mobility. The RA-induced increased RAF phosphorylation is still apparent after 72 hours of treatment when most cells are differentiated and G0 arrested. There is a progressive dose-response relationship with 10 − 8, 10 − 7, and 10 − 6 M RA. The RA-induced RAF phosphorylation corresponds to increased in vitro kinase activity. Inhibition of MEK with a PD98059 dose which inhibits ERK2 phosphorylation and subsequent cell differentiation also inhibits RAF phosphorylation. RA-induced MEK-dependent RAF phosphorylation is not due to changes in the amount of cellular MEK. The induced RAF phosphorylation, as well as anteceding ERK2 activation, depends on ligand-induced activation of both an RARα receptor and an RXR receptor. This and the slow kinetics of activation suggest a need for prior RA-induced gene expression. In summary, RA induces a MEK-dependent prolonged RAF activation, whose slow onset occurs after ERK2 activation but still well before cell cycle arrest and cell differentiation. The RA-induced increased RAF phosphorylation thus differs from typical mitogenic growth factor signaling, features that may contribute to cell cycle arrest and differentiation instead of division as the cellular outcome.

Effects of retinoids and thiazolidinediones on proliferation, insulin release, insulin mRNA, GLUT 2 transporter protein and mRNA of INS-1 cells.

Both 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (ATRA) are active metabolites of vitamin A (retinol). There exists an interaction between retinoid receptors and peroxisome proliferator-activated receptors (PPARgamma). To define their functions in an insulin secreting system the effects of ATRA, 9cRA and the PPARgamma agonist rosiglitazone on cell proliferation, insulin release and glucose transporter (GLUT) 2 of INS-1 cells were tested. Retinoic acid receptor (RAR-alpha and -gamma) and retinoid X receptor (RXR-alpha and -beta) proteins are present (immunoblots). Both 9cRA and ATRA inhibit INS-1 cell proliferation ([3H]-thymidine assay) in a concentration dependent manner. Both 9cRA and ATRA increased insulin release, but only ATRA ralsed the GLUT 2 mRNA in a bell-shaped concentration response curve after 48 h. The insulinotropic effect of one compound is not significantly superimposed by the other indicating that the same binding sites are used by 9cRA and ATRA. The acute and chronic effects of the PPARgamma agonist rosiglitazone on insulin release were additionally determined since glitazones act as transcription factors together with RXR agonists. At high concentrations (100 microM) rosiglitazone inhibited glucose (8.3 mM) stimulated insulin secretion (acute experiment over 60 min). Insulin secretion, however, was increased during a 24 h treatment at a concentration of 10 microM and again inhibited at 100 microM. Changes in preproinsulin mRNA expression were not observed. Rosiglitazone (100 microM) increased GLUT 2 mRNA paralleled by an increase of GLUT 2 protein, but only after 24 h of treatment. This data indicate that RAR and RXR mediate insulin release. The changes in GLUT 2 have no direct impact on insulin release; the inhibition seen at high concentrations of either compound is possibly the result of the observed inhibition of cell proliferation. Effects of rosiglitazone on preproinsulin mRNA and GLUT 2 (mRNA and protein) do not play a role in modulating insulin secretion. With the presence of an RXR receptor agonist the effect of rosiglitazone on insulin release becomes stimulatory. Thus the effects of RAR-, RXR agonists and rosiglitazone depend on their concentrations, the duration of their presence and are due to specific interactions.

Differential control of gene activity by isoforms A, B1 and B2 of the Drosophila ecdysone receptor

The steroid hormone ecdysone initiates molting and metamorphosis in Drosophila via a heterodimeric receptor consisting of EcR that binds hormone, and USP, a homolog of the vertebrate RXR receptor. EcR exists in three isoforms EcRA, EcRB1 and EcRB2 that are thought to direct specific physiological responses to ecdysone. These three isoforms differ only in their N-terminal A/B domain that implies that sequences responsible for the differential physiological effects lie within the A/B domains of the EcR isoforms. In the present study, we set out to determine the capability of the three isoforms and their A/B domains to control gene transcription. When full-length EcR plasmids were cotransfected into mammalian cells with a USP expressing and a cognate reporter plasmid, the three EcR isoforms showed striking differences in their ability to control gene transcription, both in the presence and in the absence of hormone. Furthermore, the A/B domains of EcRB1 and of EcRB2 when fused to the GAL4 DNA binding domain are sufficient to activate transcription of a reporter gene, in yeast as well as in mammalian cells. In contrast, a fusion construct containing the A/B domain of EcRA represses basal transcription of the reporter gene. All these findings emphasize the importance of the A/B domains of the three EcR isoforms for differentially controlling gene transcription. Furthermore, they provide evidence for the existence of an autonomous ligand-independent activation function (AF1) in the A/B domains of EcRB1 and EcRB2 and of an inhibitory function (IF) in the A/B domain of EcRA.