title: Identification of compounds that modulate retinol signaling using a cell-based qHTS assay.
abstract: In vertebrates, the retinol (vitamin A) signaling pathway (RSP) controls the biosynthesis and catabolism of all-trans retinoic acid (atRA), which regulates transcription of genes essential for embryonic development. Chemicals that interfere with the RSP to cause abnormal intracellular levels of atRA are potential developmental toxicants. To assess chemicals for the ability to interfere with retinol signaling, we have developed a cell-based RARE (Retinoic Acid Response Element) reporter gene assay to identify RSP disruptors. To validate this assay in a quantitative high-throughput screening (qHTS) platform, we screened the Library of Pharmacologically Active Compounds (LOPAC) in both agonist and antagonist modes. The screens detected known RSP agonists, demonstrating assay reliability, and also identified novel RSP agonists including kenpaullone, niclosamide, PD98059 and SU4312, and RSP antagonists including Bay 11-7085, LY294002, 3,4-Methylenedioxy-β-nitrostyrene, and topoisomerase inhibitors (camptothecin, topotecan, amsacrine hydrochloride, and idarubicin). When evaluated in the P19 pluripotent cell, these compounds were found to affect the expression of the Hoxa1 gene that is essential for embryo body patterning. These results show that the RARE assay is an effective qHTS approach for screening large compound libraries to identify chemicals that have the potential to adversely affect embryonic development through interference with retinol signaling.
authors.affiliation: Division of Molecular Biology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel, MD 20708, United States. Electronic address: yanling.chen@fda.hhs.gov.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Chen
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Srilatha
authors.initials: S
authors.lastname: Sakamuru
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Ruili
authors.initials: R
authors.lastname: Huang
authors.affiliation: Division of Molecular Biology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel, MD 20708, United States.
authors.forename: David H
authors.initials: DH
authors.lastname: Reese
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Menghang
authors.initials: M
authors.lastname: Xia
mesh_terms: D000818:Animals; D002460:Cell Line; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D008156:Luciferases / Q000235:genetics; D051379:Mice; D020218:Response Elements; D015398:Signal Transduction; D014801:Vitamin A / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D014801:Vitamin A; D008156:Luciferases
keywords: Developmental toxicant; High-throughput screening; LOPAC; Retinol signaling; Stem cell; Tox21
references.citation: Biochem J. 2002 Feb 1;361(Pt 3):621-7
references.pmid: 11802792
references.citation: Toxicol In Vitro. 2014 Mar;28(2):282-91
references.pmid: 24287113
references.citation: Annu Rev Nutr. 2001;21:167-92
references.pmid: 11375434
references.citation: Blood. 2008 Nov 1;112(9):3762-71
references.pmid: 18495959
references.citation: Leukemia. 2006 Jun;20(6):941-51
references.pmid: 16617325
references.citation: J Lipid Res. 2002 Nov;43(11):1773-808
references.pmid: 12401878
references.citation: Cancer Res. 2007 Dec 1;67(23):11386-92
references.pmid: 18056466
references.citation: Biochem Pharmacol. 2007 Aug 15;74(4):601-11
references.pmid: 17601492
references.citation: Apoptosis. 2006 Jul;11(7):1101-9
references.pmid: 16699956
references.citation: Cell Res. 2003 Dec;13(6):429-42
references.pmid: 14728799
references.citation: Oncogene. 2010 Feb 25;29(8):1135-44
references.pmid: 19935721
references.citation: J Nutr. 1995 Oct;125(10):2483-92
references.pmid: 7562082
references.citation: Dev Biol. 2001 Sep 1;237(1):107-15
references.pmid: 11518509
references.citation: Biochim Biophys Acta. 2015 Jan;1851(1):66-75
references.pmid: 24768681
references.citation: J Cell Biochem. 2009 Mar 1;106(4):682-92
references.pmid: 19160421
references.citation: J Bone Miner Metab. 2013 Jan;31(1):53-63
references.pmid: 23014973
references.citation: Cell Mol Biol Res. 1994;40(4):297-312
references.pmid: 7866431
references.citation: J Cell Physiol. 1992 May;151(2):239-48
references.pmid: 1572900
references.citation: Hum Reprod Update. 2011 Sep-Oct;17(5):589-604
references.pmid: 21747128
references.citation: J Cell Biol. 1982 Aug;94(2):253-62
references.pmid: 7107698
references.citation: Ann Hematol. 2014 Sep;93(9):1621-3
references.pmid: 24399307
references.citation: J Biomol Screen. 1999;4(2):67-73
references.pmid: 10838414
references.citation: Stem Cells. 2008 Jul;26(7):1858-64
references.pmid: 18436859
references.citation: J Am Acad Dermatol. 2004 Jun;50(6):900-6
references.pmid: 15153892
references.citation: Physiol Rev. 2000 Jul;80(3):1021-54
references.pmid: 10893430
references.citation: Sci Rep. 2014;4:5664
references.pmid: 25012808
references.citation: Trends Pharmacol Sci. 2010 Jan;31(1):36-45
references.pmid: 19896224
references.citation: Cell. 2008 Sep 19;134(6):921-31
references.pmid: 18805086
references.citation: Cell. 2006 Aug 25;126(4):663-76
references.pmid: 16904174
references.citation: J Biol Chem. 1993 Jan 5;268(1):591-600
references.pmid: 8380169
references.citation: Nat Rev Mol Cell Biol. 2015 Feb;16(2):110-23
references.pmid: 25560970
references.citation: Trends Biochem Sci. 2006 May;31(5):268-75
references.pmid: 16603362
references.citation: Annu Rev Pharmacol Toxicol. 1999;39:399-430
references.pmid: 10331090
references.citation: Leuk Res. 1994 Apr;18(4):299-304
references.pmid: 8170172
references.citation: Cancer Res. 2010 Mar 15;70(6):2516-27
references.pmid: 20215516
references.citation: Birth Defects Res B Dev Reprod Toxicol. 2013 Jun;98(3):276-82
references.pmid: 23696197
references.citation: Nucl Recept Signal. 2009;7:e002
references.pmid: 19381305
references.citation: Cancer Res. 1999 Jun 1;59(11):2566-9
references.pmid: 10363974
references.citation: J Biol Chem. 2014 Jan 10;289(2):1142-50
references.pmid: 24265316
references.citation: Physiol Genomics. 2012 May 1;44(9):495-503
references.pmid: 22433785
references.citation: Fed Proc. 1976 May 1;35(6):1332-8
references.pmid: 770206
references.citation: Dev Biol. 1999 Sep 15;213(2):390-404
references.pmid: 10479456
references.citation: Br J Pharmacol. 2013 Mar;168(5):1201-14
references.pmid: 23062100
references.citation: Sarcoma. 2012;2012:479712
references.pmid: 22701331
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11473-8
references.pmid: 16864780
references.citation: Environ Health Perspect. 2009 May;117(5):685-95
references.pmid: 19479008
references.citation: J Biol Chem. 2012 May 18;287(21):17530-45
references.pmid: 22474287
references.citation: Cell Stem Cell. 2013 Jun 6;12(6):713-26
references.pmid: 23602540
references.citation: Environ Health Perspect. 2011 Aug;119(8):1142-8
references.pmid: 21543282
references.citation: Cell Signal. 2010 Aug;22(8):1175-84
references.pmid: 20206686
references.citation: Mol Cancer. 2010;9:15
references.pmid: 20102612
references.citation: Biochemistry. 2009 Nov 3;48(43):10267-74
references.pmid: 19772353
references.citation: Dev Growth Differ. 2014 Sep;56(7):518-25
references.pmid: 25212816
references.citation: Cardiovasc Res. 2003 May 1;58(2):292-302
references.pmid: 12757864
references.citation: Neurotoxicology. 2005 Aug;26(4):531-45
references.pmid: 16112319
references.citation: Cell. 2004 Feb 20;116(4):499-509
references.pmid: 14980218
references.citation: Pharmacol Biochem Behav. 2014 May;120:117-23
references.pmid: 24582848
references.citation: Biochemistry. 2012 Feb 28;51(8):1730-9
references.pmid: 22304499
references.citation: Biochim Biophys Acta. 2002 Apr 1;1596(1):156-62
references.pmid: 11983430
references.citation: Nature. 1994 Oct 6;371(6497):528-31
references.pmid: 7935766
references.citation: J Cell Biochem. 2011 Oct;112(10):2865-72
references.pmid: 21618588
references.citation: FASEB J. 1996 Jul;10(9):940-54
references.pmid: 8801176
references.citation: Expert Opin Pharmacother. 2001 Mar;2(3):491-505
references.pmid: 11336601
references.citation: Nat Genet. 2005 Oct;37(10):1035-7
references.pmid: 16155570
references.citation: Cancer Res. 1998 Jul 15;58(14):3163-72
references.pmid: 9679985
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):8912-7
references.pmid: 19447925
references.citation: Nat Med. 2009 Apr;15(4):401-9
references.pmid: 19252500
references.citation: Cell. 2014 Mar 27;157(1):255-66
references.pmid: 24679540
references.citation: Cell Mol Life Sci. 2015 Apr;72(8):1559-76
references.pmid: 25558812
references.citation: Environ Health Perspect. 2015 Jan;123(1):49-56
references.pmid: 25302578
references.citation: Drug Discov Today. 2013 Aug;18(15-16):716-23
references.pmid: 23732176
references.citation: Biochim Biophys Acta. 2012 Jan;1821(1):152-67
references.pmid: 21621639
references.citation: ACS Med Chem Lett. 2010 Sep 07;1(9):454-9
references.pmid: 24900231
references.citation: Mol Cell Endocrinol. 2007 Jan 29;264(1-2):74-81
references.pmid: 17101211
references.citation: PLoS One. 2014;9(5):e96512
references.pmid: 24788806
references.citation: Chem Pharm Bull (Tokyo). 1999 Dec;47(12):1778-86
references.pmid: 10748721
references.citation: Cancer Res. 1973 Dec;33(12):3231-8
references.pmid: 4357355
references.citation: Cell Growth Differ. 2001 Apr;12(4):193-200
references.pmid: 11331248
references.citation: J Cell Sci. 2004 Nov 15;117(Pt 24):5731-7
references.pmid: 15522889
references.citation: Cell. 2008 Feb 8;132(3):344-62
references.pmid: 18267068
references.citation: Curr Chem Genomics. 2009 Jan 1;3(1):7-12
references.pmid: 19936037
references.citation: Cancer Lett. 2014 Jul 10;349(1):8-14
references.pmid: 24732808