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

Abstract

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.