The activation of RARα prevents surgery-induced cognitive impairments via the inhibition of neuroinflammation and the restoration of synaptic proteins in elderly mice.

Post-operative cognitive dysfunction (POCD) is a multi-etiological symptom mainly occurred in elderly people after surgery. The activation of retinoic acid receptor α (RARα), a transcriptional factor, was previously predicated to be negatively associated with the occurrence of POCD. However, the mechanisms underlying anti-POCD effects of RARα were still unclear. In this study, AM580, a selective agonist of RARα, and all-trans-retinoic acid (ATRA), a pan agonist of RAR, significantly alleviated cognitive dysfunction and increased the expression of RARα in elderly mice after surgery, which was decreased by RO41-5253, an antagonist of RARα. A bioinformatic study further predicted that the activation of RARα might produce anti-POCD effects via the restoration of synaptic proteins. Both agonists inhibited the expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (Myd88) and the phosphorylation of nuclear factorkappa-B (NF-κB), leading to the prevention of microglial over-activation and pro-inflammatory cytokines secretion in the hippocampal regions of elderly mice after surgery. Moreover, AM580 and ATRA increased the expression of brain-derived neurotrophic factor (BDNF) and postsynaptic density protein 95 (PSD95), and the phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP-response element binding protein (CREB). All these results suggested that the activation of RARα prevented surgery-induced cognitive impairments via the inhibition of neuroinflammation by the reduction of the TLR4/Myd88/NF-κB pathway and the restoration of synaptic proteins by the activation of the BDNF/ERK/CREB pathway, providing a further support that RARα could be developed as a therapeutic target for POCD.

Convergence of distinct signaling pathways on synaptic scaling to trigger rapid antidepressant action.

Ketamine is a noncompetitive glutamatergic N-methyl-d-aspartate receptor (NMDAR) antagonist that exerts rapid antidepressant effects. Preclinical studies identify eukaryotic elongation factor 2 kinase (eEF2K) signaling as essential for the rapid antidepressant action of ketamine. Here, we combine genetic, electrophysiological, and pharmacological strategies to investigate the role of eEF2K in synaptic function and find that acute, but not chronic, inhibition of eEF2K activity induces rapid synaptic scaling in the hippocampus. Retinoic acid (RA) signaling also elicits a similar form of rapid synaptic scaling in the hippocampus, which we observe is independent of eEF2K functioni. The RA signaling pathway is not required for ketamine-mediated antidepressant action; however, direct activation of the retinoic acid receptor α (RARα) evokes rapid antidepressant action resembling ketamine. Our findings show that ketamine and RARα activation independently elicit a similar form of multiplicative synaptic scaling that is causal for rapid antidepressant action.

Inhibition of epithelial-mesenchymal transition in retinal pigment epithelial cells by a retinoic acid receptor-α agonist.

Epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells plays a key role in proliferative retinal diseases such as age-related macular degeneration by contributing to subretinal fibrosis. To investigate the potential role of retinoic acid receptor-α (RAR-α) signaling in this process, we have now examined the effects of the RAR-α agonist Am580 on EMT induced by transforming growth factor-ß2 (TGF-ß2) in primary mouse RPE cells cultured in a three-dimensional type I collagen gel as well as on subretinal fibrosis in a mouse model. We found that Am580 inhibited TGF-ß2-induced collagen gel contraction mediated by RPE cells. It also attenuated the TGF-ß2-induced expression of the mesenchymal markers α-smooth muscle actin, fibronectin, and collagen type I; production of pro-matrix metalloproteinase 2 and interleukin-6; expression of the focal adhesion protein paxillin; and phosphorylation of SMAD2 in the cultured RPE cells. Finally, immunofluorescence analysis showed that Am580 suppressed both the TGF-ß2-induced translocation of myocardin-related transcription factor-A (MRTF-A) from the cytoplasm to the nucleus of cultured RPE cells as well as subretinal fibrosis triggered by laser-induced photocoagulation in a mouse model. Our observations thus suggest that RAR-α signaling inhibits EMT in RPE cells and might attenuate the development of fibrosis associated with proliferative retinal diseases.

Recent advances in the design of RAR α and RAR ß agonists as orally bioavailable drugs. A review.

Retinoic acid receptors (RARs) α, ß, and γ are members of the nuclear receptor superfamily. Compounds which bind to and activate the RARs are termed retinoids which regulate a wide variety of biological processes such as vertebrate embryonic morphogenesis and organogenesis, cell growth arrest, differentiation, and apoptosis, as well as their disorders. Although many synthetic selective RARα, RARß, and RARγ agonists have been designed and prepared, these have generally been lipophilic acids without good drug-like properties and with low oral bioavailability. Recently this has been changing and drug design approaches to highly potent and selective RARα and RARß agonists with low lipophilicity that are orally bioavailable and less toxic have been developed, that have a range of potential therapeutic uses. This review covers these new advances.

Hemodynamics-Based Strategy of Using Retinoic Acid Receptor and Retinoid X Receptor Agonists to Induce MicroRNA-10a and Inhibit Atherosclerotic Lesion.

The protocols in this chapter describe methods for identifying the functional roles of retinoic acid receptor (RAR) and retinoid X receptor (RXR) signaling in atherosclerosis and developing RARα/RXRα-specific agonists as hemodynamics-based therapeutic components for atherosclerosis treatment. In vitro cell culture flow system is used to elucidate the effects of different flow patterns and shear stresses, i.e., atherogenic oscillatory shear stress (OS) vs. atheroprotective pulsatile shear stress (PS), on RAR/RXR signaling and inflammatory responses in vascular endothelial cells (ECs). Western blotting, nuclear and cytoplasmic protein extraction, immunoprecipitation, and in situ proximity ligation assay are used to examine the expression, location, and association of RARs (i.e., RARα, RARß, and RARγ) and RXRs (i.e., RXRα, RXRß, and RXRγ) in ECs in response to OS vs. PS. Chromatin immunoprecipitation is used to examine the binding activity of RARα/RA-responsive elements (RARE). RT-microRNA (miR) quantitative real-time PCR and RT-PCR are used to detect the expressions of miR-10a and pro-inflammatory molecules, respectively. Specific siRNAs of RARα and RXRα, precursor miR-10a (PreR-10a), and antagomiR-10a (AMR-10a) are used to elucidate the regulatory roles of RARα, RXRα, and miR-10a in pro-inflammatory signaling in ECs. RARα/RXRα-specific agonists are used to induce miR-10a expression and inhibit OS-induced pro-inflammatory signaling in ECs in vitro. Apolipoprotein E-deficient (ApoE-/-) mice are used as an atherosclerotic animal model. Administration of ApoE-/- mice with RARα/RXRα-specific agonists results in inhibitions in atherosclerotic lesion formation. Co-administration of ApoE-/- mice with RARα/RXRα agonists and AMR-10a is performed to identify the role of miR-10a in RARα/RXRα agonists-mediated inhibition in atherosclerotic lesions. Oil Red O staining and H&E staining are used to examine the levels of atherosclerotic lesions in the vessel wall. In situ miR hybridization and immunohistochemical staining are used to detect the expression of miR-10a and pro-inflammatory molecules and the infiltration of inflammatory cells in the vessel wall. RARα/RXRα-specific agonists are used to mimic the atheroprotective effects of PS to induce endothelial miR-10a and hence repress OS-induced pro-inflammatory signaling and atherosclerotic lesion formation in vivo. The results indicate that RAR/RXR-specific agonists have great potential to be developed as hemodynamics-based therapeutic components for atherosclerosis treatment.

In Silico Identification and Experimental Validation of (-)-Muqubilin A, a Marine Norterpene Peroxide, as PPARα/γ-RXRα Agonist and RARα Positive Allosteric Modulator.

The nuclear receptors (NRs) RARα, RXRα, PPARα, and PPARγ represent promising pharmacological targets for the treatment of neurodegenerative diseases. In the search for molecules able to simultaneously target all the above-mentioned NRs, we screened an in-house developed molecular database using a ligand-based approach, identifying (-)-Muqubilin (Muq), a cyclic peroxide norterpene from a marine sponge, as a potential hit. The ability of this compound to stably and effectively bind these NRs was assessed by molecular docking and molecular dynamics simulations. Muq recapitulated all the main interactions of a canonical full agonist for RXRα and both PPARα and PPARγ, whereas the binding mode toward RARα showed peculiar features potentially impairing its activity as full agonist. Luciferase assays confirmed that Muq acts as a full agonist for RXRα, PPARα, and PPARγ with an activity in the low- to sub-micromolar range. On the other hand, in the case of RAR, a very weak agonist activity was observed in the micromolar range. Quite surprisingly, we found that Muq is a positive allosteric modulator for RARα, as both luciferase assays and in vivo analysis using a zebrafish transgenic retinoic acid (RA) reporter line showed that co-administration of Muq with RA produced a potent synergistic enhancement of RARα activation and RA signaling.

Correction of Defective T-Regulatory Cells From Patients With Crohn's Disease by Ex Vivo Ligation of Retinoic Acid Receptor-α.

BACKGROUND & AIMS: Crohn's disease (CD) is characterized by an imbalance of effector and regulatory T cells in the intestinal mucosa. The efficacy of anti-adhesion therapies led us to investigate whether impaired trafficking of T-regulatory (Treg) cells contributes to the pathogenesis of CD. We also investigated whether proper function could be restored to Treg cells by ex vivo expansion in the presence of factors that activate their regulatory activities. METHODS: We measured levels of the integrin α4ß7 on Treg cells isolated from peripheral blood or lamina propria of patients with CD and healthy individuals (controls). Treg cells were expanded ex vivo and incubated with rapamycin with or without agonists of the retinoic acid receptor-α (RARA), and their gene expression profiles were analyzed. We also studied the cells in cytokine challenge, suppression, and flow chamber assays and in SCID mice with human intestinal xenografts. RESULTS: We found that Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. The pathway that regulates the expression of integrin subunit α is induced by retinoic acid (RA). Treg cells from patients with CD incubated with rapamycin and an agonist of RARA (RAR568) expressed high levels of integrin α4ß7, as well as CD62L and FOXP3, compared with cells incubated with rapamycin or rapamycin and all-trans retinoic acid. These Treg cells had increased suppressive activities in assays and migrated under conditions of shear flow; they did not produce inflammatory cytokines, and RAR568 had no effect on cell stability or lineage commitment. Fluorescently labeled Treg cells incubated with RAR568 were significantly more likely to traffic to intestinal xenografts than Treg cells expanded in control medium. CONCLUSIONS: Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. Incubation of patients' ex vivo expanded Treg cells with rapamycin and an RARA agonist induced expression of α4ß7 and had suppressive and migratory activities in culture and in intestinal xenografts in mice. These cells might be developed for treatment of CD. ClinicalTrials.gov, Number: NCT03185000.

Ro41-5253, a selective antagonist of retinoic acid receptor α, ameliorates chronic unpredictable mild stress-induced depressive-like behaviors in rats: Involvement of regulating HPA axis and improving hippocampal neuronal deficits.

Accumulating reports have highlighted an association between excess retinoids and depression development. Retinoic acid receptor α (RARα) is implicated in the activation of hypothalamus-pituitary-adrenal (HPA) axis and closely involved in the etiology of depression, suggesting it might be a novel target of antidepressant. This study investigated the antidepressant potential of Ro41-5253 (a selective RARα antagonist) and related mechanisms using a depression rat model imitated by social isolation and chronic unpredicted mild stress (CUMS). Sucrose preference test (SPT), open field test (OFT) and forced swimming test (FST) were applied to assess the antidepressant-like effect. HPA axis activity, RARα expression in hypothalamic paraventricular nucleus (PVN) and hypothalamus, and protein levels of brain-derived neurotrophic factor (BDNF) and synapse-related proteins (PSD95, SYP, MAP2) in hippocampus were measured, respectively. We found that Ro41-5253 treatment ameliorated the depressive-like behaviors in CUMS rats, as evidenced by increased sucrose preference in SPT, raised numbers of crossing and rearing in OFT, reduced immobility time and prolonged swimming time in FST. The HPA axis hyperactivity was attenuated by Ro41-5253 (1 mg/kg) treatment, indicated by reduced serum corticosterone level, decreased adrenal gland index, reduced corticotrophin-releasing hormone protein level in hypothalamus, and recovered hypothalamic glucocorticoid receptor protein level. In addition, Ro41-5253 (1 mg/kg) treatment downregulated RARα protein expression in hypothalamic PVN and hypothalamus, and increased the protein levels of BDNF, PSD95, SYP and MAP2 in the hippocampus. We concluded that Ro41-5253 had antidepressant-like effects on CUMS rats by downregulating HPA axis hyperactivity and improving the hippocampal neuronal deficits.

All-trans retinoic acid suppresses bone morphogenetic protein 4 in mouse diabetic nephropathy through a unique retinoic acid response element.

Diabetic nephropathy (DN) causes mesangial matrix expansion, which results in glomerulosclerosis and renal failure. Collagen IV (COL4) is a major component of the mesangial matrix that is positively regulated by bone morphogenetic protein 4 (BMP4)/suppressor of mothers against decapentaplegic (Smad1) signaling. Because previous studies showed that retinoids treatment had a beneficial effect on kidney disease, we investigated the therapeutic potential of retinoids in DN, focusing especially on the regulatory mechanism of BMP4. Diabetes was induced with streptozotocin in 12-wk-old male Crl:CD1(ICR) mice, and, 1 mo later, we initiated intraperitoneal injection of all-trans retinoic acid (ATRA) three times weekly. Glomerular matrix expansion, which was associated with increased BMP4, phosphorylated Smad1, and COL4 expression, worsened in diabetic mice at 24 wk of age. ATRA administration alleviated DN and downregulated BMP4, phosopho-Smad1, and COL4. In cultured mouse mesangial cells, treatment with ATRA or a retinoic acid receptor-α (RARα) agonist significantly decreased BMP4 and COL4 expression. Genomic analysis suggested two putative retinoic acid response elements (RAREs) for the mouse Bmp4 gene. Chromatin immunoprecipitation analysis and reporter assays indicated a putative RARE of the Bmp4 gene, located 11,488-11,501 bp upstream of exon 1A and bound to RARα and retinoid X receptor (RXR), which suppressed BMP4 expression after ATRA addition. ATRA suppressed BMP4 via binding of a RARα/RXR heterodimer to a unique RARE, alleviating glomerular matrix expansion in diabetic mice. These findings provide a novel regulatory mechanism for treatment of DN.

2-Bromopalmitate targets retinoic acid receptor alpha and overcomes all-trans retinoic acid resistance of acute promyelocytic leukemia.

Fatty acid oxidation dependency of leukemia cells has been documented in recent studies. Pharmacologic inhibition of fatty acid oxidation, thereby, displays significant effects in suppressing leukemia. 2-Bromopalmitate, a palmitate analogue, was initially identified as an inhibitor of fatty acid oxidation, and recently recognized as an inhibitor of protein palmitoylation. However, the effects of 2-Bromopalmitate on leukemia and its cellular targets remain obscure. Herein, we discover in cultured cell lines, a transplantable mouse model, and primary blasts that 2-Bromopalmitate presents synergistic differentiation induction with all-trans retinoic acid in acute promyelocytic leukemia. Moreover, 2-Bromopalmitate overcomes all-trans retinoic acid resistance in all-trans retinoic acid-resistant cells and leukemic mice. Mechanistically, 2-Bromopalmitate covalently binds at cysteine 105 and cysteine 174 of retinoic acid receptor alpha (RARα) and stabilizes RARα protein in the presence of all-trans retinoic acid which is known to induce RARα degradation, leading to enhanced transcription of RARα-target genes. Mutation of both cysteines largely abrogates the synergistic effect of 2-Bromopalmitate on all-trans retinoic acid-induced differentiation, demonstrating that 2-Bromopalmitate promotes all-trans retinoic acid-induced differentiation through binding RARα. All-trans retinoic acid-based regimens including arsenic trioxide or chemotherapy, as preferred therapy for acute promyelocytic leukemia, induce adverse events and irreversible resistance. We expect that combining all-trans retinoic acid with 2-Bromopalmitate would be a promising therapeutic strategy for acute promyelocytic leukemia, especially for overcoming all-trans retinoic acid resistance of relapsed acute promyelocytic leukemia patients.

Induction of microRNA-10a using retinoic acid receptor-α and retinoid x receptor-α agonists inhibits atherosclerotic lesion formation.

BACKGROUND AND AIMS: MicroRNA (miR)-10a is a shear-regulated miR with the lowest expression in vascular endothelial cells (ECs) in athero-susceptible regions with oscillatory shear stress (OS). The aim of this study is to elucidate the relationship between EC miR-10a and atherosclerosis and develop a hemodynamics-based strategy for atherosclerosis treatment. METHODS: A combination of in vitro flow system and in vivo experimental animals was used to examine the functional roles of EC miR-10a and its clinical applications in atherosclerosis. RESULTS: En face staining showed that EC miR-10a is down-regulated in the inner curvature (OS region) of aortic arch in rats. Co-administration with retinoic acid receptor-α (RARα)- and retinoid X receptor-α (RXRα)-specific agonists rescued EC miR-10a expression in this OS region. These effects of OS and RARα/RXRα-specific agonists on EC miR-10a expression were confirmed by the in vitro flow system, and were modulated by the RARα-histone deacetylases complex, with the consequent modulation in the downstream GATA6/vascular cell adhesion molecule (VCAM)-1 signaling cascade. Animal studies showed that miR-10a levels are decreased in both aortic endothelium of atherosclerotic lesions and blood plasma from apolipoprotein E-deficient (ApoE-/-) mice. In vivo induction of EC miR-10a by administration of RARα/RXRα-specific agonists protects ApoE-/- mice from atherosclerosis through inhibition of GATA6/VCAM-1 signaling and inflammatory cell infiltration. CONCLUSIONS: Our findings indicate that down-regulation of miR-10a in aortic endothelium and blood serum is associated with atherosclerosis, and miR-10a has potential to be developed as diagnostic molecule for atherosclerosis. Moreover, EC miR-10a induction by RARα/RXRα-specific agonists is a potential hemodynamics-based strategy for atherosclerosis treatment.

Activation of RARα, RARγ, or RXRα Increases Barrier Tightness in Human Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells.

The blood-brain barrier (BBB) is critical to central nervous system (CNS) health. Brain microvascular endothelial cells (BMECs) are often used as in vitro BBB models for studying BBB dysfunction and therapeutic screening applications. Human pluripotent stem cells (hPSCs) can be differentiated to cells having key BMEC barrier and transporter properties, offering a renewable, scalable source of human BMECs. hPSC-derived BMECs have previously been shown to respond to all-trans retinoic acid (RA), and the goal of this study was to identify the stages at which differentiating human induced pluripotent stem cells (iPSCs) respond to activation of RA receptors (RARs) to impart BBB phenotypes. Here the authors identified that RA application to iPSC-derived BMECs at days 6-8 of differentiation led to a substantial elevation in transendothelial electrical resistance and induction of VE-cadherin expression. Specific RAR agonists identified RARα, RARγ, and RXRα as receptors capable of inducing barrier phenotypes. Moreover, RAR/RXRα costimulation elevated VE-cadherin expression and improved barrier fidelity to levels that recapitulated the effects of RA. This study elucidates the roles of RA signaling in iPSC-derived BMEC differentiation, and identifies directed agonist approaches that can improve BMEC fidelity for drug screening studies while also distinguishing potential nuclear receptor targets to explore in BBB dysfunction and therapy.

Design and synthesis of a potent, highly selective, orally bioavailable, retinoic acid receptor alpha agonist.

A ligand-based virtual screening exercise examining likely bioactive conformations of AM 580 (2) and AGN 193836 (3) was used to identify the novel, less lipophilic RARα agonist 4-(3,5-dichloro-4-ethoxybenzamido)benzoic acid 5, which has good selectivity over the RARß, and RARγ receptors. Analysis of the medicinal chemistry parameters of the 3,5-substituents of derivatives of template 5 enabled us to design a class of drug-like molecules with lower intrinsic clearance and higher oral bioavailability which led to the novel RARα agonist 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 that has high RARα potency and excellent selectivity versus RARß (2 orders of magnitude) and RARγ (4 orders of magnitude) at both the human and mouse RAR receptors with improved drug-like properties. This RARα specific agonist 56 has high oral bioavailability (>80%) in both mice and dogs with a good PK profile and was shown to be inactive in cytotoxicity and genotoxicity screens.

Retinoic acid induction of CD1d expression primes chronic lymphocytic leukemia B cells for killing by CD8+ invariant natural killer T cells.

Invariant natural killer T (iNKT) cells are cytotoxic T cells that respond to glycolipid antigens presented by CD1d. Therapeutic activation of iNKT cells with α-galactosylceramide (α-GalCer) can prevent and reverse tumor growth in mice and clinical trials involving α-GalCer-stimulated iNKT cells are ongoing in humans. B cells express CD1d, however, we show that CD1d expression is reduced on B cells from patients with chronic lymphocytic leukemia (CLL). B cells from CLL patients pulsed with α-GalCer failed to stimulate cytolytic degranulation by iNKT cell lines, but could present the more potent glycolipid analogue, 7DW8-5. Retinoic acid receptor-α (RAR-α) agonists induced CD1d expression by CLL B cells, restoring their ability to present α-GalCer to CD8α+ iNKT cells, resulting in cytolytic degranulation. Thus, RAR-α agonists can augment the anti-tumor activities of iNKT cells against CLL cells in vitro. Their inclusion in iNKT cell-based therapies may benefit patients with CLL.

A Novel Role for RARα Agonists as Apolipoprotein CIII Inhibitors Identified from High Throughput Screening.

Elevated triglyceride (TG) levels are well-correlated with the risk for cardiovascular disease (CVD). Apolipoprotein CIII (ApoC-III) is a key regulator of plasma TG levels through regulation of lipolysis and lipid synthesis. To identify novel regulators of TG levels, we carried out a high throughput screen (HTS) using an ApoC-III homogenous time resolved fluorescence (HTRF) assay. We identified several retinoic acid receptor (RAR) agonists that reduced secreted ApoC-III levels in human hepatic cell lines. The RARα specific agonist AM580 inhibited secreted ApoC-III by >80% in Hep3B cells with an EC50 ~2.9 nM. In high-fat diet induced fatty-liver mice, AM580 reduced ApoC-III levels in liver as well as in plasma (~60%). In addition, AM580 treatment effectively reduced body weight, hepatic and plasma TG, and total cholesterol (TC) levels. Mechanistically, AM580 suppresses ApoC-III synthesis by downregulation of HNF4α and upregulation of SHP1 expression. Collectively, these studies suggest that an RARα specific agonist may afford a new strategy for lipid-lowering and CVD risk reduction.

Superenhancer Analysis Defines Novel Epigenomic Subtypes of Non-APL AML, Including an RARα Dependency Targetable by SY-1425, a Potent and Selective RARα Agonist.

We characterized the enhancer landscape of 66 patients with acute myeloid leukemia (AML), identifying 6 novel subgroups and their associated regulatory loci. These subgroups are defined by their superenhancer (SE) maps, orthogonal to somatic mutations, and are associated with distinct leukemic cell states. Examination of transcriptional drivers for these epigenomic subtypes uncovers a subset of patients with a particularly strong SE at the retinoic acid receptor alpha (RARA) gene locus. The presence of a RARA SE and concomitant high levels of RARA mRNA predisposes cell lines and ex vivo models to exquisite sensitivity to a selective agonist of RARα, SY-1425 (tamibarotene). Furthermore, only AML patient-derived xenograft (PDX) models with high RARA mRNA were found to respond to SY-1425. Mechanistically, we show that the response to SY-1425 in RARA-high AML cells is similar to that of acute promyelocytic leukemia treated with retinoids, characterized by the induction of known retinoic acid response genes, increased differentiation, and loss of proliferation.Significance: We use the SE landscape of primary human AML to elucidate transcriptional circuitry and identify novel cancer vulnerabilities. A subset of patients were found to have an SE at RARA, which is predictive for response to SY-1425, a potent and selective RARα agonist, in preclinical models, forming the rationale for its clinical investigation in biomarker-selected patients. Cancer Discov; 7(10); 1136-53. ©2017 AACR.See related commentary by Wang and Aifantis, p. 1065.This article is highlighted in the In This Issue feature, p. 1047.

The change in retinoic acid receptor signaling induced by prenatal marginal vitamin A deficiency and its effects on learning and memory.

This study investigated the prenatal marginal vitamin A deficiency (mVAD)-related impairment in learning and memory and the interactions between RARα, Src and NR1. Learning and memory were assessed in adult rats that were exposed to prenatal mVAD with Morris water maze. The average escape time was longer in mVAD rats, and they passed the hidden platform fewer times during the memory retention test than normal vitamin A intake (VAN) rats. The mRNA and protein levels of RARα, Src and NR1 in mVAD rats were significantly lower than those in VAN rats. RARα and Src, but not NR1, were in the same protein complex. RA treatment-induced increase in RARα, Src and NR1 expressions in mVAD neurons was much lower than that in VAN neurons. Overexpression of RARα gene in VAN neurons induced an increase in RARα, Src and NR1 expressions, while silencing of RARα gene induced a decrease in expressions of RARα and Src, but not that of of NR1. In mVAD neurons, however, overexpression of RARα did not induce an increase in NR1 expression, while silencing of RARα gene had no effect on Src and NR1 expressions. Furthermore, inhibition of Src was associated with a decrease in NR1 expression but not that of RARα. Prenatal mVAD was associated with impaired learning and memory in adult rats. It is possible that mVAD-related decrease in RARα led to a decrease in Src expression, which in turn down-regulated NR1 expression and Ca2+ influx and eventually caused learning and memory deficits.

Characterization of the differential coregulator binding signatures of the Retinoic Acid Receptor subtypes upon (ant)agonist action.

Retinoic Acid Receptor alpha (RARα/NR1B1), Retinoic Acid Receptor beta (RARß/NR1B2) and Retinoic Acid Receptor gamma (RARγ/NR1B3) are transcription factors regulating gene expression in response to retinoids. Within the RAR genomic pathways, binding of RARs to coregulators is a key intermediate regulatory phase. However, ligand-dependent interactions between the wide variety of coregulators that may be present in a cell and the different RAR subtypes are largely unknown. The aim of this study is to characterize the coregulator binding profiles of RARs in the presence of the pan-agonist all-trans-Retinoic Acid (AtRA); the subtype-selective agonists Am80 (RARα), CD2314 (RARß) and BMS961 (RARγ); and the antagonist Ro415253. To this end, we used a microarray assay for coregulator-nuclear receptor interactions to assess RAR binding to 154 motifs belonging to >60 coregulators. The results revealed a high number of ligand-dependent RAR-coregulator interactions among all RAR variants, including many binding events not yet described in literature. Next, this work confirmed a greater ligand-independent activity of RARß compared to the other RAR subtypes based on both higher basal and lower ligand-driven coregulator binding. Further, several coregulator motifs showed selective binding to a specific RAR subtype. Next, this work showed that subtype-selective agonists can be successfully discriminated by using coregulator binding assays. Finally this study demonstrated the possible applications of a coregulator binding assay as a tool to discriminate between agonistic/antagonistic actions of ligands. The RAR-coregulator interactions found will be of use to direct further studies to better understand the mechanisms driving the eventual actions of retinoids.