The endogenous retinoid metabolite S-4-oxo-9-cis-13,14-dihydro-retinoic acid activates retinoic acid receptor signalling both in vitro and in vivo.
Retinoic acid receptor (RAR) and retinoid X receptor are ligand-induced transcription factors that belong to the nuclear receptor family. The receptors are activated by small hydrophobic compounds, such as all-trans-retinoic acid and 9-cis-retinoic acid, respectively. Interestingly, these receptors are also targets for a number of exogenous compounds. In this study, we characterized the biological activity of the 9-cis-substituted retinoic acid metabolite, S-4-oxo-9-cis-13,14-dihydro-retinoic acid (S-4o9cDH-RA). The endogenous levels of this metabolite in wild-type mice and rats were found to be higher than those of all-trans-retinoic acid, especially in the liver. Using cell-based luciferase reporter systems, we showed that S-4o9cDH-RA activates the transcription of retinoic acid response element-containing genes in several cell types, both from a simple 2xDR5 element and from the promoter of the natural retinoid target gene RARbeta2. In addition, quantitative RT-PCR analysis demonstrated that S-4o9cDH-RA treatment significantly increases the endogenous mRNA levels of the RAR target gene RARbeta2. Utilizing a limited proteolytic digestion assay, we showed that S-4o9cDH-RA induces conformational changes to both RARalpha and RARbeta in the same manner as does all-trans-retinoic acid, suggesting that S-4o9cDH-RA is indeed an endogenous ligand for these receptors. These in vitro results were corroborated in an in vivo system, where S-4o9cDH-RA induced morphological changes similar to those of all-trans-retinoic acid in the developing chicken wing bud. When locally applied to the wing bud, S-4o9cDH-RA induced digit pattern duplications in a dose-dependent fashion. The results illustrate that S-4o9cDH-RA closely mimics all-trans-retinoic acid with regard to pattern respecification. Finally, using quantitative RT-PCR analysis, we showed that S-4o9cDH-RA induces the transcription of several retinoic acid-regulated genes in chick wing buds, including Hoxb8, RARbeta2, shh, Cyp26 and bmp2. Although S-4o9cDH-RA was less potent when compared with all-trans-retinoic acid, the findings clearly demonstrate that S-4o9cDH-RA has the capacity to bind and activate nuclear retinoid receptors and regulate gene transcription both in vitro and in vivo.
Schuchardt
Jan P
JP
Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine, Hannover, Germany. jan-philipp.schuchardt@lw.uni-hannover.de
Wahlström
David
D
Rüegg
Joëlle
J
Giese
Norbert
N
Stefan
Madalina
M
Hopf
Henning
H
Pongratz
Ingemar
I
Håkansson
Helen
H
Eichele
Gregor
G
Pettersson
Katarina
K
Nau
Heinz
H
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002642:Chick Embryo; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D018878:Limb Buds / Q000187:drug effects / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D015394:Molecular Structure; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000072482:Retinoic Acid Receptor alpha; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D014212:Tretinoin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D014921:Wings, Animal / Q000187:drug effects / Q000196:embryology
C464754:9-cis-4-oxo-13,14-dihydro-retinoic acid; C000607071:RARA protein, human; C000607072:Rara protein, mouse; C000607073:Rara protein, rat; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D000072482:Retinoic Acid Receptor alpha; C082925:retinoic acid receptor beta; D014212:Tretinoin; D008156:Luciferases