Retinoic Acid Receptor Pathway Research Articles

The Endogenous Retinoic Acid Receptor Pathway Is Exploited by Mycobacterium tuberculosis during Infection, Both InVitro and InVivo.

Retinoic acid (RA) is a fundamental vitamin A metabolite involved in regulating immune responses through the nuclear RA receptor (RAR) and retinoid X receptor. While performing experiments using THP-1 cells as a model for Mycobacterium tuberculosis infection, we observed that serum-supplemented cultures displayed high levels of baseline RAR activation in the presence of live, but not heat-killed, bacteria, suggesting that M. tuberculosis robustly induces the endogenous RAR pathway. Using invitro and invivo models, we have further explored the role of endogenous RAR activity in M. tuberculosis infection through pharmacological inhibition of RARs. We found that M. tuberculosis induces classical RA response element genes such as CD38 and DHRS3 in both THP-1 cells and human primary CD14+ monocytes via a RAR-dependent pathway. M. tuberculosis-stimulated RAR activation was observed with conditioned media and required nonproteinaceous factor(s) present in FBS. Importantly, RAR blockade by (4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthalen-2-yl]ethenyl]benzoic acid), a specific pan-RAR inverse agonist, in a low-dose murine model of tuberculosis significantly reduced SIGLEC-F+CD64+CD11c+high alveolar macrophages in the lungs, which correlated with 2× reduction in tissue mycobacterial burden. These results suggest that the endogenous RAR activation axis contributes to M. tuberculosis infection both invitro and invivo and reveal an opportunity for further investigation of new antituberculosis therapies.

Stimulation of the farnesoid X receptor promotes M2 macrophage polarization.

FXR is a key molecule that modulates anti-inflammatory activity in the intestinal-liver axis. Although FXR has pleiotropic functions including regulation of liver inflammation and activation of macrophages, it remains unclear whether it is involved in macrophage polarization. In this paper we demonstrated that stimulation of macrophages derived from the bone marrow using an FXR agonist activated polarization toward M2 but not M1 macrophages. The treatment of mice with chitin skewed macrophage polarization towards M2 macrophages, while co-treatment with an FXR agonist further promoted the polarization toward M2 macrophages in vivo. This skewed polarization towards M2 macrophages by an FXR agonist was accompanied by increased expression of signaling molecules related to the retinoic acid receptor. Inhibition of the retinoic acid receptor suppressed FXR agonist-mediated M2 macrophage polarization, indicating that this polarization was, at least, partly dependent on the retinoic acid receptor pathway. These data demonstrate that FXR has a role in polarization toward M2 macrophages and suggest a possible therapeutic potential of FXR agonists in M2 macrophage-related conditions.

Abstract 1519: ALDH1A1 enhances micro homology-mediated end joining by increasing POLQ expression

Abstract Aldehyde dehydrogenase (ALDH) is a superfamily of 19 known enzymes that participated in the metabolism of endogenous and exogenous aldehydes. ALDH1A1 is a major subtype contributing to high ALDH activity out of all ALDH isoforms and plays a critical role in the maintenance of cancer stem cells. Our previous studies have shown that ALDH1A1 is able to reduce the sensitivity of BRCA2-/- ovarian cancer cells to PARP inhibitors by enhancing microhomology-mediated end joining (MMEJ). MMEJ uses 5-25 base pair micro homologous sequences to align the broken strands before joining, and this repair pathway requires DNA polymerase θ (Pol θ, encoded by POLQ). Here we show that ALDH1A1 can increase the expression of Pol θ, at both mRNA and protein levels, in ovarian cancer cell lines. ALDH1A1 is reported to catalyze the production of all-trans retinoic acid (ATRA), which can bind to the retinoic acid receptor (RAR), and promote transcription of the downstream target genes that possess retinoic acid response element (RARE) in their promoter regions. Interestingly we identified several RAREs in the promoter region of the POLQ gene. Thus, we hypothesized that ALDH1A1 promotes Pol θ expression via activation of the RAR signaling pathway. We found that ATRA and RAR agonist (CH55) increased but pan RAR antagonist (AGN193109) treatment decreased the expression of POLQ, indicating that the RAR pathway is involved in the transcription of POLQ. We also demonstrated that RARα can bind to the promoter region of Pol Q, activation of RAR signaling by treatment with ATRA or CH55 increased the enrichment of K27 acetylated histone H3 in this promoter region. Moreover, we showed that mutated ALDH1A1 lacking the enzyme activity was unable to enhance POLQ transcription. These data suggest that ALDH1A1 enhances the Pol Q expression by increasing the production of ATRA which further activates the RAR signaling pathway. Citation Format: Kousalya Lavudi, Ananya Banerjee, Shurui Cai, Na Li, Xuetao Bai, Wang Qi-En. ALDH1A1 enhances micro homology-mediated end joining by increasing POLQ expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1519.

Attenuation of Hypertrophy in Human MSCs via Treatment with a Retinoic Acid Receptor Inverse Agonist.

In vitro chondrogenically differentiated mesenchymal stem cells (MSCs) have a tendency to undergo hypertrophy, mirroring the fate of transient “chondrocytes” in the growth plate. As hypertrophy would result in ossification, this fact limits their use in cartilage tissue engineering applications. During limb development, retinoic acid receptor (RAR) signaling exerts an important influence on cell fate of mesenchymal progenitors. While retinoids foster hypertrophy, suppression of RAR signaling seems to be required for chondrogenic differentiation. Therefore, we hypothesized that treatment of chondrogenically differentiating hMSCs with the RAR inverse agonist, BMS204,493 (further named BMS), would attenuate hypertrophy. We induced hypertrophy in chondrogenic precultured MSC pellets by the addition of bone morphogenetic protein 4. Direct activation of the RAR pathway by application of the physiological RAR agonist retinoic acid (RA) further enhanced the hypertrophic phenotype. However, BMS treatment reduced hypertrophic conversion in hMSCs, shown by decreased cell size, number of hypertrophic cells, and collagen type X deposition in histological analyses. BMS effects were dependent on the time point of application and strongest after early treatment during chondrogenic precultivation. The possibility of modifing hypertrophic cartilage via attenuation of RAR signaling by BMS could be helpful in producing stable engineered tissue for cartilage regeneration.

Fatty Acid Binding Protein FABP5: A Novel Therapeutic Target in Acute Myeloid Leukemia

Introduction: Retinoic acid (RA), the active metabolite of vitamin A, influences biological processes by activating the retinoic acid receptor (RAR). RARs are ligand-controlled transcription factors that function as heterodimer with retinoid X receptors (RXRs) and regulate expression of target genes affecting homeostasis of cellular differentiation and death. The success of RAR activation in the treatment of acute promyelocytic leukemia (APL), particularly by the use of all-trans retinoic acid (ATRA) has stimulated considerable interest in the development of small molecules that can modulate RAR and RXR in AML cells. RA can also activate the pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ) in a context dependent manner. In the aqueous intracellular milieu, RA is transported by the cellular retinoid-binding protein CRABP-II, or by the fatty-acid-binding protein FABP5, depending on the ratio of FABP5 to CRABP-II and cellular levels of RA. In cells, expressing high CRABP-II and low FABP5, RA activates the RAR, whereas in the presence of the reverse ratio, RA activates PPARβ/δ. These two different modes of RA delivery leads to opposite cellular outcomes. Cells harboring high level of CRABP-II, RA is delivered to RAR leading to differentiation and growth arrest. However, FABP5 high expressing cells, RA is delivered to PPARβ/δ resulting in survival and proliferation. Therefore, preventing FABP5 from such antagonism may be a novel therapeutic strategy for AML with high FABP5. Here, we report the development of a therapeutic approach using a specific FABP5 inhibitor (iFABP5) that facilitates the delivery of RA to tumor suppressor RAR. Methods: Genetic as well as pharmacologic models in AML cell lines were used to validate the therapeutic target by decoupling PPARβ/δ and RAR/RXR pathways. Knockdown and overexpression of key regulators of these pathways were rationally used to understand pro-survival effects of FABP5 overexpression. The efficacy of small molecule pharmacophore were evaluated in vitro in cell free and cell culture model systems as well as in vivo small animal model systems as a proof of therapeutic concept. Results: We analyzed AML samples from different cohorts (Beat AML and TCGA) for the expression levels of genes involved in RAR and PPARβ/δ pathways. Most of the AML patients have upregulation of pro-survival PPARβ/δ pathway genes including FABP5 and downregulation of differentiating promoting RAR pathway genes including critical RA chaperone CRABP2. On the otherhand, ATRA sensitive APL patients and cell lines have lower expression of FABP5 compared to AML. We established FABP5 as a therapeutic target for ATRA sensitization in AML cell line THP1. Genetic ablation of FABP5 using shRNA sensitizes THP1 cells to ATRA treatment compared to scramble control cells. We used in silico approach to develop a novel small molecule iFABP5 that binds to recombinant FABP5 (Kd= 2x10-7) with a 56-fold higher affinity compared to ATRA (Kd=11x10-6 Mole) in a cell free fluorescence reporter binding assay using 8-Anilino-1-naphthalenesulfonic acid (ANS). Efficacy of iFABP5 as a single agent and in combination with ATRA was evaluated in THP1, HL-60, K562, and OCI-AML5 cells. The effect of the combination was most pronounced in THP-1 cells. The iFABP5 synergizes with ATRA and induces the differentiation at 72 hours as assessed by both CD11b and CD14 levels. Treatment of cells with single agent iFABP5 leads to downregulation of PPARβ/δ target genes and upregulation of RAR target genes consistent with the genetic ablation of shFABP5. THP1 cells stably expressing shRNA targeting FABP5 has significant growth perturbation in a NSG mouse model compared to non-targeting shRNA. Pre-clinical in vivo evaluation of iFABP5 in combination with ATRA is ongoing as a therapeutic proof of concept. Conclusion: The lack of effective ATRA response in AML might be due to an aberrant activation of pro-survival PPARβ/δ pathway, which negatively affects ATRA-regulated gene expression and its antileukemic activity. Reprogramming of the RA delivery to RAR pathway with the addition of a small molecular inhibitor of FABP5 could potentially restore therapeutic effects of ATRA in FABP5 over expressing cases of AML subtypes. Disclosures Maciejewski: Novartis: Consultancy; Alexion: Consultancy.

β-Cryptoxanthin Induces UCP-1 Expression via a RAR Pathway in Adipose Tissue

While β-cryptoxanthin is hypothesized to have a preventive effect on lifestyle-related diseases, its underlying mechanisms are unknown. We investigated the effect of β-cryptoxanthin on energy metabolism in adipose tissues and its underlying mechanism. C57BL/6J mice were fed a high-fat diet (60% kcal fat) containing 0 or 0.05% β-cryptoxanthin for 12 weeks. β-cryptoxanthin treatment was found to reduce body fat gain and plasma glucose level, while increasing energy expenditure. The expression of uncoupling protein (UCP) 1 was elevated in adipose tissues in the treatment group. Furthermore, the in vivo assays showed that the Ucp1 mRNA expression was higher in the β-cryptoxanthin treatment group, an effect that disappeared upon cotreatment with a retinoic acid receptor (RAR) antagonist. In conclusion, we report that β-cryptoxanthin reduces body fat and body weight gain and that β-cryptoxanthin increases the expression of UCP1 via the RAR pathway.

The retinoic acid receptor (RAR) α-specific agonist Am80 (tamibarotene) and other RAR agonists potently inhibit hepatitis B virus transcription from cccDNA

Chronic infection with the human Hepatitis B virus (HBV) is a major global health problem. Hepatitis D virus (HDV) is a satellite of HBV that uses HBV envelope proteins for cell egress and entry. Using infection systems encoding the HBV/HDV receptor human sodium taurocholate co-transporting polypeptide (NTCP), we screened 1181 FDA-approved drugs applying markers for interference for HBV and HDV infection. As one primary hit we identified Acitretin, a retinoid, as an inhibitor of HBV replication and HDV release. Based on this, other retinoic acid receptor (RAR) agonists with different specificities were found to interfere with HBV replication, verifying that the retinoic acid receptor pathway regulates replication. Of the eight agonists investigated, RARα-specific agonist Am80 (tamibarotene) was most active. Am80 reduced secretion of HBeAg and HBsAg with IC50s < 10 nM in differentiated HepaRG-NTCP cells. Similar effects were observed in primary human hepatocytes. In HepG2-NTCP cells, profound Am80-mediated inhibition required prolonged treatment of up to 35 days. Am80 treatment of cells with an established HBV cccDNA pool resulted in a reduction of secreted HBsAg and HBeAg, which correlated with reduced intracellular viral RNA levels, but not cccDNA copy numbers. The effect lasted for >12 days after removal of the drug. HBV genotypes B, D, and E were equally inhibited. By contrast, Am80 did not affect HBV replication in transfected cells or HepG2.2.15 cells, which carry an integrated HBV genome. In conclusion, our results indicate a persistent inhibition of HBV transcription by Am80, which might be used for drug repositioning.

Recurrent RARB Translocations in Acute Promyelocytic Leukemia Lacking RARA Translocation.

Translocations of retinoic acid receptor-α (RARA), typically PML-RARA, are a genetic hallmark of acute promyelocytic leukemia (APL). However, because a small fraction of APL lack translocations of RARA, we focused here on APL cases without RARA translocation to elucidate the molecular etiology of RARA-negative APL. We performed whole-genome sequencing, PCR, and FISH for five APL cases without RARA translocations. Four of five RARA-negative APL cases had translocations involving retinoic acid receptor-β (RARB) translocations, and TBL1XR1-RARB was identified as an in-frame fusion in three cases; one case had an RARB rearrangement detected by FISH, although the partner gene could not be identified. When transduced in cell lines, TBL1XR1-RARB homodimerized and diminished transcriptional activity for the retinoic acid receptor pathway in a dominant-negative manner. TBL1XR1-RARB enhanced the replating capacity of mouse bone marrow cells and inhibited myeloid maturation of human cord blood cells as PML-RARA did. However, the response of APL with RARB translocation to retinoids was attenuated compared with that of PML-RARA, an observation in line with the clinical resistance of RARB-positive APL to ATRA. Our results demonstrate that the majority of RARA-negative APL have RARB translocations, thereby forming a novel, distinct subgroup of APL. TBL1XR1-RARB as an oncogenic protein exerts effects similar to those of PML-RARA, underpinning the importance of retinoic acid pathway alterations in the pathogenesis of APL.Significance: These findings report a novel and distinct genetic subtype of acute promyelocytic leukemia (APL) by illustrating that the majority of APL without RARA translocations harbor RARB translocations. Cancer Res; 78(16); 4452-8. ©2018 AACR.

MP81-14 EXOSOMES SECRETED BY PLACENTAL STEM CELLS SELECTIVELY INHIBIT GROWTH OF PROSTATE CANCER CELLS

You have accessJournal of UrologyStem Cell Research: Stem Cell Research II1 Apr 2018MP81-14 EXOSOMES SECRETED BY PLACENTAL STEM CELLS SELECTIVELY INHIBIT GROWTH OF PROSTATE CANCER CELLS Taylor Peak, Prakash Praharaj, Gati Panigrahi, Yixin Su, Isabel Schlaepfer, Ravi Singh, Donald Vander Griend, Julie Allickson, Anthony Atala, and Gagan Deep Taylor PeakTaylor Peak More articles by this author , Prakash PraharajPrakash Praharaj More articles by this author , Gati PanigrahiGati Panigrahi More articles by this author , Yixin SuYixin Su More articles by this author , Isabel SchlaepferIsabel Schlaepfer More articles by this author , Ravi SinghRavi Singh More articles by this author , Donald Vander GriendDonald Vander Griend More articles by this author , Julie AllicksonJulie Allickson More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and Gagan DeepGagan Deep More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2722AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES For patients who develop castrate-resistant prostate cancer (CRPC), enzalutamide represents an androgen deprivation therapy (ADT) utilized by clinicians. However, as with other forms of ADT, patients become resistant to enzalutamide, prompting the need to identify agents with a novel mechanism of action. We identified exosomes derived from c-kit selected placental stem cells (PLSC) as a therapeutic option for CRPC. METHODS We isolated exosomes from conditioned media of PLSC by ultracentrifugation and characterized size distribution and number by nanoparticle tracking analysis, as well as protein and lipid content by Mass Spectrometry. We treated enzalutamide-sensitive(S) and resistant(R) C4-2B, CWR-R1, LNCaP cells, as well as non-neoplastic prostate (PREC-1 and PWR-R1) and fibroblast (M3T3) cells with PLSC exosomes. We tested the cytotoxicity of exosomes on all cell lines by MTT Assay. We then performed a clonogenic assay using the same dose of PLSC exosomes on C4-2B-S, C4-2B-R, and PWR-R1. Finally, to understand the mechanism behind their biological efficacy, we treated C4-2B cells with exosomes and examined changes in protein expression. RESULTS PLSC exosomes were loaded with high concentrations of saturated long chain fatty acids (LCFAs) (A). These exosomes inhibited cell viability of all prostate cancer cells lines by 30-50%. In addition, there was no effect on the viability of non-neoplastic cells. PLSC exosomes significantly inhibited clone formation in C4-2B-sensitive and resistant cells by 47% and 64%, respectively (B). There was no inhibition of clone formation in the non-neoplastic PWR-R1 cell line (C). Based on our protein and lipid data of PLSC exosomes, we explored the retinoic acid receptor pathway as a potential mechanism of action. We found that after treating C4-2B cells with exosomes, there was a significant increase in expression of retinoic acid receptor γ. CONCLUSIONS PLSC exosomes selectively inhibit growth of enzalutamide-sensitive and resistant cell lines, without affecting the growth of non-cancerous cells. This growth inhibition is associated with an increase in retinoid acid receptor γ, a known tumor suppressor that is regulated by LCFAs. We propose that the LCFAs loaded in exosomes activate retinoid acid receptor γ, thus suppressing the growth of prostate cancer tumor cells. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1102 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Taylor Peak More articles by this author Prakash Praharaj More articles by this author Gati Panigrahi More articles by this author Yixin Su More articles by this author Isabel Schlaepfer More articles by this author Ravi Singh More articles by this author Donald Vander Griend More articles by this author Julie Allickson More articles by this author Anthony Atala More articles by this author Gagan Deep More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Inhibition Of LSD1 As a Therapeutic Strategy For The Treatment Of Acute Myeloid Leukemia

Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. These complexes include Histone Deacetylases (HDAC1/2) and Co-Repressor for Element-1-Silencing Transcription factor (CoREST). LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression. LSD1 has been shown to play a role in development in various contexts. LSD1 can interact with pluripotency factors in human embryonic stem cells and is important for decommissioning enhancers in stem cell differentiation. Beyond embryonic settings, LSD1 is also critical for hematopoietic differentiation. LSD1 is overexpressed in multiple cancer types and recent studies suggest inhibition of LSD1 reactivates the all-trans retinoic acid receptor pathway in acute myeloid leukemia (AML). These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and through its presence in transcriptional complexes.The current study describes the anti-tumor effects of a novel LSD1 inhibitor (GSK2879552) in AML. GSK2879552 is a potent, selective, mechanism-based, irreversible inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML cells have a unique requirement for LSD1. While LSD1 inhibition did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of putative LSD1 target genes. This increase in the transcriptionally activating histone modification correlated with a dose dependent increase in gene expression. Treatment with GSK2879552 promoted the expression of cell surface markers, including CD11b and CD86, associated with a differentiated immunophenotype in 12 of 13 AML cell lines. For example, in SKM-1 cells, increases in cell surface expression of CD86 and CD11b occurred after as early as one day of treatment with EC50 values of 13 and 7 nM respectively. In a separate study using an MV-4-11 engraftment model, increases in CD86 and CD11b were observed as early as 8 hours post dosing.GSK2879552 treatment resulted in a potent anti-proliferative growth effect in 19 of 25 AML cell lines (average EC50 = 38 nM), representing a range of AML subtypes. Potent growth inhibition was also observed on AML blast colony forming ability in 4 out of 5 bone marrow samples derived from primary AML patient samples (average EC50 = 205 nM). The effects of LSD1 inhibition were further characterized in an in vivo mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and upon engraftment, the mice were treated for 17 days. After 17 days of treatment, control treated mice had 80% GFP+ cells in the bone marrow whereas treated mice possessed 2.8% GFP positive cells (p<0.012). The percentage of GFP+ cells continued to decrease to 1.8% by 1-week post therapy. Remarkably, in a preliminary assessment for survival, control-treated mice succumbed to AML by 28 days post transplant, while treated mice showed prolonged survival.Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. GSK2879552 is currently in late preclinical development and clinical trials are anticipated to start in 2014.All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Disclosures:Kruger:GlaxoSmithKline Pharmaceuticals: Employment. Mohammad:GlaxoSmithKline Pharmaceuticals: Employment. Smitheman:GlaxoSmithKline Pharmaceuticals: Employment. Liu:GlaxoSmithKline Pharmaceuticals: Employment. Pappalardi:GlaxoSmithKline Pharmaceuticals: Employment. Federowicz:GlaxoSmithKline Pharmaceuticals: Employment. Van Aller:GlaxoSmithKline Pharmaceuticals: Employment. Kasparec:GlaxoSmithKline Pharmaceuticals: Employment. Tian:GlaxoSmithKline Pharmaceuticals: Employment. Suarez:GlaxoSmithKline Pharmaceuticals: Employment. Rouse:GlaxoSmithKline Pharmaceuticals: Employment. Schneck:GlaxoSmithKline Pharmaceuticals: Employment. Carson:GlaxoSmithKline Pharmaceuticals: Employment. McDevitt:GlaxoSmithKline Pharmaceuticals: Employment. Ho:GlaxoSmithKline Pharmaceuticals: Employment. McHugh:GlaxoSmithKline Pharmaceuticals: Employment. Miller:GlaxoSmithKline Pharmaceuticals: Employment. Johnson:GlaxoSmithKline Pharmaceuticals: Employment. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other. Tummino:GlaxoSmithKline Pharmaceuticals: Employment.

Activation of a retinoic acid receptor pathway by thiazolidinediones induces production of vascular endothelial growth factor/vascular permeability factor in OP9 adipocytes

Thiazolidinediones, ligands of peroxisome proliferator-activated receptorγ (PPARγ), are used in the management of type 2 diabetes mellitus. However, they can cause edema, which often leads to a discontinuation of treatment. The mechanism by which thiazolidinediones induce edema is poorly understood. We have confirmed that troglitazone (TGZ), a thiazolidinedione, induced the differentiation of a preadipocyte cell line, OP9, into adipocytes. The differentiated OP9 cells produced vascular permeability factors and the activity was completely neutralized by an antibody against vascular endothelial growth factor (VEGF). TGZ induced the expression of VEGF but not interleukin-6 and monocyte chemoattractant protein-1. 2-chloro-5-nitrobenzanilide (GW9662) blocked both the differentiation and the production of VEGF induced by TGZ. 15-deoxy-Δ12,14-Prostaglandin J2, a natural ligand of PPARγ, and another PPARγ agonist, ginkgolic acid, also induced an increase in the expression of VEGE as well as the differentiation of OP9 cells. Indomethacin, a nonsteroidal anti-inflammatory drug (NSAID) with PPARγ activity, up-regulated VEGF expression, but acetylsalicylic acid, a NSAID without PPARγ activity, did not. Although VEGF expression was enhanced under hypoxic conditions, the expression of hypoxia inducible factor and Ets-1 was down-regulated during the TGZ-induced differentiation. On the other hand, retinoic acid enhanced the expression of VEGF despite inhibiting the TGZ-induced differentiation. Moreover, retinoic acid receptor (RAR) β expression was increased by TGZ and retinoic acid. These findings suggested that the major adipocyte-derived vascular permeability factor produced in response to TGZ was VEGF, and a RAR pathway was involved in the production.

Abstract A28: The link between the metabolic effects of nuclear receptor (NR) modulators and anticarcinogenic properties on leukemia cancer cells can be revealed through studying the Warburg effect

Abstract The Warburg effect is the observation that most cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by aerobic oxidation of pyruvate in mitochondria. Genetic studies in humans and rodents support the notion that metabolic enzymes controlling glucose metabolism can directly contribute to carcinogenesis. Current knowledge of nuclear receptor (NR) signaling networks indicates that NRs control a wide variety of metabolic processes by regulating the expression of genes encoding key enzymes, transporters, and other proteins involved in metabolic homeostasis. Thus, a better understanding of the mechanistic links between cellular metabolism control by NRs and cancer cells' growth control may ultimately lead to better treatments for human cancer. We created a library of 134 NR modulators including compounds targeting the estrogen receptor (ER), androgen receptor (AR), farnesoid X receptor (FXR), peroxisome proliferator-activated receptor (PPAR), retinoic acid receptor (RAR), retinoid X receptor (RXR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), liver X receptor (LXR), thyroid receptor (TR), and pregnane X receptor (PXR). We used high-throughput assays to measure cell viability, apoptosis, and cell proliferation of leukemic cell lines after treatment with this library. Afterwards, compounds which highly induced apoptosis and inhibited cell proliferation were picked for further investigation of their mechanism of action. We linked the effect of those compounds on apoptosis to their effect on lactate levels. In addition, we tested the effect of these compounds on the activity of pyruvate dehydrogenase complex (PDC) which catalyzes the irreversible oxidative decarboxylation of pyruvate into acetyl-CoA an important step in commitment of glucose into full oxidation rather than glycolysis, PDH dipstick assay was used for this purpose. Of the 134 nuclear receptors modulators that we have tested, nine compounds induced significant apoptosis in Jurkat cells. These compounds mainly targeted ER and RAR. On the other hand, only six compounds from this list significantly reduced the lactate levels in Jurkat cells. Finally, the compounds modulating the RAR pathway were the only compounds that showed significant effects on PDC activity. This work may open a door to further explore the RAR modulators as strong potential anticarcinogenic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A28.

Retinoic acid induces expression of Ig germ line α transcript, an IgA isotype switching indicative, through retinoic acid receptor

Retinoic acid (RA) is considered to possess an activity of IgA isotype switching. Thus far, TGF-β1 is known to be the most powerful IgA isotype switch factor. To elucidate the molecular mechanisms underlying the Ig germ line (GL) α transcriptional regulation by RA, we constructed three different sizes of mouse GLα promoter reporters; short-GLα(−130/+14), middle-GLα(−448/+72) and long-GLα(−3028/+72). Based on luciferase assay, RA increased the activity of all three GLα promoter reporters by approximately 2-fold and the effect was further enhanced by TGF-β1. Overexpression of Smad3/4 increased TGF-β1-induced GLα promoter activities but had no effect on RA-induced GLα promoter activities. In order to analyze the characteristics of the RA-inducible GLα promoter region, we also constructed two mutant reporters: Smad3 binding elements (SBEs)-substituted short-GLα (short-GLα mSBE) and Runx3 binding elements (RBEs)-substituted short-GLα (short-GLα mRBE) promoter reporters. Promoter activities of the two mutant reporters to RA were comparable to that of wild type reporter, while those of the two mutant reporters to TGF-β1 were markedly diminished as compared to that of WT short-GLα. Finally, RA-induced GLα transcription was virtually disappeared by LE540, an antagonist of RA receptor (RAR). Taken together, these results suggest that RA induces GLα transcription mainly through RAR pathway, where neither Smad3/4 nor Runx3 is involved.

Hexadecanoic Acid from Buzhong Yiqi Decoction Induced Proliferation of Bone Marrow Mesenchymal Stem Cells

Buzhong Yiqi decoction (BYD) is a well-known ancient tonic prescription in traditional Chinese medicine (TCM). The purpose of this study is to identify active components of BYD involved in promoting proliferation of mesenchymal stem cells (MSCs) and to investigate its mechanism. BYD was extracted with petroleum ether, ethanol, and water. Evidence provided by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, bromodeoxyuridine, proliferation cell nuclear antigen immunoreactivity, cell cycle analysis, and gas chromatography-mass spectrometry indicated that hexadecanoic acid (HA) in BYD extracted with petroleum ether is the active compound responsible for increasing proliferation of MSCs. Western blot analysis show that HA significantly increase retinoic acid receptor (RAR) levels of MSCs, but not estrogen receptor, thyroid hormone receptor, vitamin D receptor, glucocorticoid receptor, and peroxisome proliferator-activated receptor. Reverse transcription-polymerase chain reaction revealed that HA significantly increased RAR mRNA levels. Furthermore, the mechanism of HA action depends on RAR pathway and up-regulates expression of mRNA for insulin-like growth factor-I, the target gene of RAR. Our findings have now allowed for a refinement in our understanding of TCM with respect to pharmacological regulation of stem cells and may be useful to stem cell biology and therapy.

Trialkyltin Compounds Bind Retinoid X Receptor to Alter Human Placental Endocrine Functions

Retinoid X receptor (RXR) is a nuclear receptor that plays important and multiple roles in mammalian development and homeostasis. We previously reported that, in human choriocarcinoma cells, tributyltin chloride and triphenyltin hydroxide, which are typical environmental contaminants and cause masculinization in female mollusks, are potent stimulators of human chorionic gonadotropin production and aromatase activity, which play key endocrine functions in maintaining pregnancy and fetal development. However, the molecular mechanism through which these compounds stimulate these endocrine functions remains unclear. Our current study shows that trialkyltin compounds, including tributyltin chloride and triphenyltin hydroxide, function as RXR agonists. Trialkyltins directly bind to the ligand-binding domain of RXR with high affinity and function as transcriptional activators. Unlike the natural RXR ligand, 9-cis-retinoic acid, the activity of trialkyltins is RXR specific and does not activate the retinoic acid receptor pathway. In addition, trialkyltins activate RXR to stimulate the expression of a luciferase reporter gene containing the human placental promoter I.1 sequence of aromatase, suggesting that trialkyltins stimulate human placental endocrine functions through RXR-dependent signaling pathways. Therefore, our results suggest that activation of RXR may be a novel mechanism by which trialkyltins alter human endocrine functions.

Retinoic acid isomers produce malformations in postembryonic development of the Japanese flounder, Paralichthys olivaceus.

We previously reported that characteristic deformities were induced by retinoic acid (RA) treatment of the Japanese flounder, Paralichthys olivaceus, at 6-9 days post-hatching (dph). To evaluate the toxic potency of nuclear retinoid receptors in induction of deformities by RA, we here investigated the effects of retinoic acid isomers on postembryonic development of this species. Larvae were exposed to either 25 nM of all-trans RA (atRA), 9-cis RA (9cRA) or 13-cis RA (13cRA) at 6-9 dph. All RA isomers induced deformities in the lower jaw, caudal fin and vertebrae. In the lower jaw, growth retardation of the dentary was evident. In the vertebrae, the major abnormalities were hypertrophy of the centrum, central fusion, and an increase in the number of abdominal vertebrae. Caudal fin deformities included deformity of caudal bone complex and absence of the entire caudal fin. The absence of the hypural primordium at 12 dph was the first sign of abnormality in caudal fin development, and resulted in complete blocking of the caudal fin development. Among the RA isomers, atRA induced the most severe deformity in all skeletons examined. Retinoic acid receptor (RAR) expression was activated by atRA and 9cRA, and pitx2 expression was inhibited in the lower jaw by atRA and 9cRA. Vitamin D receptor (VDR) expression was specifically inhibited by atRA treatment, suggesting that RA inhibits the lower jaw growth by suppressing the expression of these genes. These results suggest that RA exerted toxic effects on the skeletal systems, mainly through the RAR pathway.

Induction of apoptosis in naive mouse T cells upon peroxisome proliferator-activated receptor gamma (PPAR-γ) binding

Thiazolidinediones, ligands of peroxisome proliferator-activated receptorγ (PPARγ), are used in the management of type 2 diabetes mellitus. However, they can cause edema, which often leads to a discontinuation of treatment. The mechanism by which thiazolidinediones induce edema is poorly understood. We have confirmed that troglitazone (TGZ), a thiazolidinedione, induced the differentiation of a preadipocyte cell line, OP9, into adipocytes. The differentiated OP9 cells produced vascular permeability factors and the activity was completely neutralized by an antibody against vascular endothelial growth factor (VEGF). TGZ induced the expression of VEGF but not interleukin-6 and monocyte chemoattractant protein-1. 2-chloro-5-nitrobenzanilide (GW9662) blocked both the differentiation and the production of VEGF induced by TGZ. 15-deoxy-Δ12,14-Prostaglandin J2, a natural ligand of PPARγ, and another PPARγ agonist, ginkgolic acid, also induced an increase in the expression of VEGE as well as the differentiation of OP9 cells. Indomethacin, a nonsteroidal anti-inflammatory drug (NSAID) with PPARγ activity, up-regulated VEGF expression, but acetylsalicylic acid, a NSAID without PPARγ activity, did not. Although VEGF expression was enhanced under hypoxic conditions, the expression of hypoxia inducible factor and Ets-1 was down-regulated during the TGZ-induced differentiation. On the other hand, retinoic acid enhanced the expression of VEGF despite inhibiting the TGZ-induced differentiation. Moreover, retinoic acid receptor (RAR) β expression was increased by TGZ and retinoic acid. These findings suggested that the major adipocyte-derived vascular permeability factor produced in response to TGZ was VEGF, and a RAR pathway was involved in the production.

Overexpression of Wild-Type Retinoic Acid Receptor  (RAR) Recapitulates Retinoic Acid-Sensitive Transformation of Primary Myeloid Progenitors by Acute Promyelocytic Leukemia RAR-Fusion Genes

Retinoic acid receptor  (RAR) is the target of several chromosomal translocations associated with acute promyelocytic leukemias (APLs). These rearrangements fuse RAR to different partner genes creating the chimeric proteins: PML-RAR, PLZF-RAR, and NPM-RAR. Although the vast majority of APLs respond to retinoic acid therapy, those associated with PLZF-RAR are resistant. We have used retroviruses to express PML-RAR, PLZF-RAR, NPM-RAR, RAR403 (a dominant negative mutant of RAR), and wild-type RAR in murine bone marrow progenitors and found that all of these constructs blocked differentiation and led to the immortalization of myeloid progenitors. This cellular transformation is specific to an alteration of the RAR pathway because overexpression of RARβ, RARγ, or RXR did not result in similar growth perturbations. Pharmacological doses of RA induced differentiation and inhibited proliferation of cells transformed with either of the APL fusion genes, including PLZF-RAR, whereas physiological retinoic acid concentrations were sufficient to reverse the phenotype of cells transformed with wild-type RAR. The cellular responses to retinoic acid were accompanied by a sharp decrease in the amount of the RAR-fusion proteins expressed in the cells. Our findings suggest that the oncogenicity of RAR-fusion proteins results from their nature to behave as unliganded RAR in the presence of physiological concentrations of retinoic acid.

Activation of mammalian retinoid X receptors by the insect growth regulator methoprene.

We report that methoprene and its derivatives can stimulate gene transcription in vertebrates by acting through the retinoic acid-responsive transcription factors, the retinoid X receptors (RXRs). Methoprene is an insect growth regulator in domestic and agricultural use as a pesticide. At least one metabolite of methoprene, methoprene acid, directly binds to RXR and is a transcriptional activator in both insect and mammalian cells. Unlike the endogenous RXR ligand, 9-cis-retinoic acid, this activity is RXR-specific; the methoprene derivatives do not activate the retinoic acid receptor pathway. Methoprene is a juvenile hormone analog that acts to retain juvenile characteristics during insect growth, preventing metamorphosis into an adult, and it has been shown to have ovicidal properties in some insects. Thus, a pesticide that mimics the action of juvenile hormone in insects can also activate a mammalian retinoid-responsive pathway. This finding provides a basis through which the potential bioactivity of substances exposed to the environment may be reexamined and points the way for discovery of new receptor ligands in both insects and vertebrates.