Identification of natural ligands of retinoic acid receptor-related orphan receptor alpha ligand-binding domain expressed in Sf9 cells--a mass spectrometry approach

The ligand-binding domain (LBD) of the human retinoic acid receptor-related orphan receptor (RORalpha-LBD), expressed in Sf9 cells, was purified and analyzed by electrospray ionization-mass spectrometry (ESI-MS). ESI-MS operated under native conditions showed the presence of a fortuitous ligand with molecular weight 386. Further analysis by gas chromatography-mass spectrometry (GC-MS) allowed the identification of the ligands bound to the LBD. Cholesterol (77%) and 7-dehydrocholesterol (provitamin D(3); 18%) were shown to be the major ligands. A monohydroxylated cholesterol derivative was identified as a minor ligand. In addition, ligand exchange experiments monitored by ESI-MS showed that cholesterol sulfate has a higher affinity for RORalpha-LBD than cholesterol and 25-hydroxycholesterol. Binding of coactivator (CoA) peptide GRIP1P was shown to occur in a stoichiometric manner. Therefore, monitoring of binding of CoAs by mass spectrometry could be used for classification of the ligands as agonist or antagonist molecules.     

Response of human rhabdomyosarcoma cell lines to retinoic acid: relationship with induction of differentiation and retinoic acid sensitivity

The ability of retinoids to induce growth inhibition associated with differentiation of diverse cell types makes them potent anti-cancer agents. We examined the effect of retinoic acid (RA) in cell lines derived from rhabdomyosarcoma (RMS), a malignant soft-tissue tumor committed to the myogenic lineage, but arrested prior to terminal differentiation. We showed that several RMS derived cell lines, including RD human rhabdomyosarcoma cells, are resistant to the growth-inhibitory and differentiation effects of RA. We established that this RA-resistance correlates with reduced expression and activity of RA-receptors in RD cells. We stably expressed either RARalpha, RARbeta, RARgamma, or RXRalpha expression vector into RD cells and found that only RARbeta or RARgamma induced a significant RA growth arrest without promoting differentiation indicating that changes in the amounts of RARs and RXRs are not sufficient to determine the RA myogenic response of rhabdomyosarcoma cells. Activation of RD cell differentiation by ectopic MRF4 expression enhanced RA-receptor activity and led to RA induction of differentiation. These studies demonstrate that RA-resistance of RD cells is linked to their lack of differentiation and suggest that the differentiation-promoting activity of RA requires factors other than RAR-RXR heterodimers.     

Regulation of the murine alpha(2)(I) collagen promoter by retinoic acid and retinoid X receptors

Retinoic acid decreases collagen production by hepatic stellate cells. This study investigated the effects of retinoic acid receptor beta (RARbeta) and retinoid X receptor alpha (RXRalpha) on the regulation of the alpha(2)(I) collagen promoter. Retinoic acid and the RARbeta and RXRalpha expression vectors suppressed the promoter in transfected stellate cells with maximal suppression obtained when combined. Mutation of the retinoic acid response element (RARE) at -879 to -874 (site 1) enhanced promoter activity and diminished but did not eliminate the suppression by RARbeta and RXRalpha. Mutation of another RARE site (site 2), at -930 to -911, resulted in low activity that was inhibited by retinoic acid. Mutation of the AP-2-binding site enhanced promoter activity that was inhibited by retinoic acid. This study shows that the suppressive effect of retinoic acid on the promoter is maximal with a combination of RARbeta and RXRalpha and occurs at more than one RARE site. The effect of retinoic acid is not mediated by AP-2.     

Differential regulation of CC chemokine receptors by 9-cis retinoic acid in the human mast cell line, HMC-1

Mast cells are well known as effector cells in a variety of inflammatory diseases, including asthma as well as other allergic disorders. The precise role of 9-cis retinoic acid (9CRA) in mast cells is not understood despite the accepted fact that 9CRA regulates inflammatory responses and neutrophil differentiation. In this study, we investigated the effects of 9CRA on the expression of CC chemokine receptors in the human mast cell line, HMC-1. 9CRA selectively inhibits the CCR2 mRNA level and increases the CCR3 mRNA level in both a time and dose dependent manner. Other CC chemokine receptors, including CCR1, CCR4 and CCR5 are not altered by treatment with 9CRA. Both TNF-alpha and LPS, known pro-inflammatory molecules, have no effect on mRNA levels of CC chemokine receptors. For surface expression, 9CRA decreased the CCR2 level but had no effect on the CCR3 level. 9CRA inhibited the chemotactic activity in response to the CCR2-dependent chemokine, MCP-1/CCL2 but not in response to CCR3-specific chemokine, eotaxin/CCL11. 9CRA decreased spontaneous homotype clustering. Therefore, our results demonstrate that 9CRA differentially decreases both CCR2 expression and chemotactic ability of HMC-1 cells, and may regulate the inflammatory effects of mast cells.     

Effect of marginal vitamin A deficiency during pregnancy on retinoic acid receptors and N-methyl-D-aspartate receptor expression in the offspring of rats

This study examined whether pregnancy-related marginal vitamin A deficiency (MVAD) influences postnatal development of retinoic acid receptors (RARs) and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1) in hippocampus of rat pups. Sixteen female rats were randomized equally into control and MVAD groups. Dams and pups were fed with either a normal control diet or one deficient in vitamin A. Eight female pups in each group were killed at 1 day, 2 weeks, 4 weeks and 8 weeks after birth, respectively. Serum retinol levels were monitored. The messenger RNA (mRNA) and protein expressions and subcellular localization of RARα, RARβ and NR1 in postnatal hippocampus were detected. At 1 day, 2 weeks and 8 weeks after birth, serum retinol levels in the MVAD group were significantly lower than those in the control group. Results of Morris water maze test at 7 weeks of age showed that spatial learning and memory in the MVAD group were affected. Vitamin A deficiency resulted in decreased mRNA levels of RARα, RARβ and NR1 (P<.05). The protein level of RARα and NR1 in the MVAD group was lower than that of the control group (P<.05). There was no significant difference in RARβ between the groups (P>.05). A mass of RARα and NR1 colocalized in hippocampal cell cytoplasm on postnatal day 1. Our data suggested that vitamin A deficiency in pregnancy may affect the postnatal expression of RARα and NR1, affecting learning and memory function in the hippocampus and synaptic plasticity of the calcium signaling pathway.     

Targeting RORs nuclear receptors by novel synthetic steroidal inverse agonists for autoimmune disorders

Designing novel inverse agonists of NR RORγt still represents a challenge for the pharmaceutical community to develop therapeutics for treating immune diseases. By exploring the structure of NRs natural ligands, the representative arotenoid ligands and RORs specific ligands share some chemical homologies which can be exploited to design a novel molecular structure characterized by a polycyclic core bearing a polar head and a hydrophobic tail. Compound MG 2778 (8), a cyclopenta[a]phenantrene derivative, was identified as lead compound which was chemically modified at position 2 in order to obtain a small library for preliminary SARs. Cell viability and estrogenic activity of compounds 7, 8, 19a, 30, 31 and 32 were evaluated to attest selectivity. The selected 7, 8, 19a and 31 compounds were assayed in a Gal4 UAS-Luc co-transfection system in order to determine their ability to modulate RORγt activity in a cellular environment. They were evaluated as inverse agonists taken ursolic acid as reference compound. The potency of compounds was lower than that of ursolic acid, but their efficacy was similar. Compound 19a was the most active, significantly reducing RORγt activity at low micromolar concentrations.     

Identification of a decarboxylation product of retinoic acid

The oxidative decarboxylation of retinoic acid was investigated utilizing a model system concisting of all-trans-retinoic acid, H₂O₂ and horseradish peroxidase. The decarboxylation products were purified by high-performance liquid chromatography on bonded, octadecylsilane columns. Based on mass spectral, nuclear magnetic resonance, ultraviolet and Fourier transform infrared analyses, the major decarboxylation product was identified as a 4-oxo-C₁₉ aldehyde with a hyrdoxyl group on the side chain at C₉, specifically 8-(2,6,6,-trimethyl-3-oxocyclohex-1-enyl)2,6-dimethyl-6-hydroxyoctatrienal.     

9-cis-retinoic acid analogues with bulky hydrophobic rings: new RXR-selective agonists

Stille cross-coupling of aryltriflates 10 and dienylstannane 11, oxidation and Horner–Wadsworth–Emmons reaction afforded stereoselectively retinoates 15. Saponification provided the carboxylic acids 8a and 8b, retinoids that incorporate a bulky hydrophobic ring while preserving the 9-cis-geometry of the parent system. In contrast to the pan-RAR/RXR agonistic profile of the lower homologue of 8a, compound 7 (LG100567), retinoids 8 showed selective binding and transactivation of RXR, devoid of significant RAR activation. In PLB985 leukemia cells that require RXR agonists for differentiation compounds 8 induced maturation in the presence of the RAR-selective pan-agonist TTNPB; this effect was blocked by an RXR-selective antagonist.     

Nuclear all-trans retinoic acid receptors

The present study was undertaken to investigate the effects of selenite (Se^IV) and selenate (Se^VI) on the all-trans retinoic acid (RA)-nuclear retinoic acid receptor (RAR) complex formation in rat liver. We also present the data on the in vitro effects of Se^IV on the RARα and the type I iodothyronine 5′-deiodinase gene expression in the GH₄C₁ rat pituitary tumor cells. Se^IV at 1.0 μmol/L was found to reduce (p<0.05) the RA specific binding to RAR in rat liver. Dithiothreitol (DTT), a protective agent for sulfhydryl groups, was found to be slightly effective in protecting the RAR binding properties when affected by Se^IV. Se^VI at 0.1 μmol/L reduced (p<0.05) the RA specific binding to RAR in liver, as well. Seleno-l-methionine (Se-^II) when compared tol-methionine did not exert any inhibitory effect on the formation of the RA-RAR complex. Se^IV (up to 2.5 μmol/L) has no inhibitory effect on GH₄C₁ cell proliferation as well as the prolactin secretion. Se^IV at 1.0 μmol/L significantly decreases the rate of mRNA synthesis and/or degradation of the α form of the RAR and causes the enhancement of the type I iodothyronine 5′-deiodinase gene expression in GH₄C₁ cells. The results based on in vitro experiments suggest that inorganic selenium may affect the RA specific binding to their cognate receptor molecules, and it may reduce expression of the gene encoding the RARα, with the cell vitality and the cell growth remaining unchanged.     

Development of novel lithocholic acid derivatives as vitamin D receptor agonists

Lithocholic acid (2) was identified as the second endogenous ligand of vitamin D receptor (VDR), though its binding affinity to VDR and its vitamin D activity are very weak compared to those of the active metabolite of vitamin D₃, 1α,25-dihydroxyvitamin D₃ (1). 3-Acylated lithocholic acids were reported to be slightly more potent than lithocholic acid (2) as VDR agonists. Here, aiming to develop more potent lithocholic acid derivatives, we synthesized several derivatives bearing a 3-sulfonate/carbonate or 3-amino/amide substituent, and examined their differentiation-inducing activity toward human promyelocytic leukemia HL-60 cells. Introduction of a nitrogen atom at the 3-position of lithocholic acid (2) decreased the activity, but compound 6 bearing a 3-methylsulfonate group showed more potent activity than lithocholic acid (2) or its acylated derivatives. The binding of 6 to VDR was confirmed by competitive binding assay and X-ray crystallographic analysis of the complex of VDR ligand-binding domain (LBD) with 6.     

Beta-adrenergic receptors: Regulatory role of agonists

Direct radioligand binding studies have been used to probe the molecular mechanisms whereby agonist catecholamines regulate the function of betaadrenergic receptors in a model system, the frog erythrocyte. The unique characteristics of agonist as opposed to antagonist action are first, the ability to stimulate the adenylate cyclase through the receptor and second, the ability to desensitize the system by alterations induced in beta-adrenergic receptors. These properties of agonist are not shared by antagonist despite the high affinity and specificity of antagonist binding to the beta-adrenergic receptors. Agonist and antagonist receptor complexes may be distinguished in a variety of ways including differences in their sensitivity to regulatory guanine nucleotides and also by gel chromatography on AcA 34 Ultragel. The agonist receptor complex appears to elute from the columns with an apparently increased size. A “dynamic receptor affinity model” of beta-adrenergic receptor action is proposed which features several distinct conformational states of the receptor. Agonists have much higher affinity for the physiologically active or coupled state of the receptor, whereas antagonists have equal affinity for both. In addition, a third “desensitized” state of the receptor is also postulated to exist.