Novel retinoic acid receptor ligands in Xenopus embryos.

Abstract

Retinoids are a large family of natural and synthetic compounds related to vitamin A that have pleiotropic effects on body physiology, reproduction, immunity, and embryonic development. The diverse activities of retinoids are primarily mediated by two families of nuclear retinoic acid receptors, the RARs and RXRs. Retinoic acids are thought to be the only natural ligands for these receptors and are widely assumed to be the active principle of vitamin A. However, during an unbiased, bioactivity-guided fractionation of Xenopus embryos, we were unable to detect significant levels of all-trans or 9-cis retinoic acids. Instead, we found that the major bioactive retinoid in the Xenopus egg and early embryo is 4-oxoretinaldehyde, which is capable of binding to and transactivating RARs. In addition to its inherent activity, 4-oxoretinaldehyde appears to be a metabolic precursor of two other RAR ligands, 4-oxoretinoic acid and 4-oxoretinol. The remarkable increase in activity of retinaldehyde and retinol as a consequence of 4-oxo derivatization suggests that this metabolic step could serve a critical regulatory function during embryogenesis.Retinoic acid (RA) is a vitamin A metabolite that has profound effects on growth, differentiation, and development (1). RA is a potent teratogen in human fetal development (2) and influences limb development and regeneration (for reviews, see refs. 3 and 4). RA is also capable of altering positional identities along the vertebrate anteroposterior axis. Treatment of early Xenopus embryos with RA elicits concentrationdependent anterior truncations, which some have interpreted to represent posterior transformations (5). The majority of the effects of RA are likely to result from alterations in gene expression elicited by the two classes of ligand-dependent transcription factors, RA receptors (RARs) and 9-cis RA receptors (RXRs) (for review, see ref. 6).Our interest in the effects of endogenous retinoids in Xenopus development led us to undertake a search for naturally occurring compounds capable of activating RARs. In preliminary experiments, we were intrigued by the relative paucity of all-trans and 9-cis RAs compared with other RAR agonists in the egg and early embryo, leading us to look for other retinoid derivatives. Previous attempts had focused on separating embryonic extracts by HPLC and matching the retention times of components having absorbance at 350 nm with those of known standard retinoids (5, 7-9). To identify potentially novel compounds, we used HPLC fractionation and purification guided simply by biological activity as measured by transcriptional regulation. This strategy does not presume prior knowledge of endogenous retinoids and has the benefit of being able to identify compounds with unusual fractionation and absorption properties.This approach identified 4-oxoretinaldehyde (4-oxo-RAL) as the major bioactive retinoid in the Xenopus egg and early embryo. In addition to being able to bind to and transactivate RARs, 4-oxo-RAL appears to be a metabolic precursor o...

Spatiotemporal retinoid-X receptor activation detected in live vertebrate embryos

“…CYP26 expression may explain the lack of gRXR activation in the brain and the inability of exogenous retinoic acid isomers to induce reporter gene expression anterior to the spinal cord, whereas the synthetic ligand LG268 is able to do so. Although endogenous concentrations of retinoids have been measured in early X. laevis embryos (49), other studies in frogs and mice have failed to show significant levels of 9-cis RA in the developing embryo (50,51). These findings raise questions about a physiological role for endogenous 9-cis RA as a physiological RXR ligand.…”

Induction of Larval Tissue Resorption in Xenopus laevis Tadpoles by the Thyroid Hormone Receptor Agonist GC-1

“…Protease protection assays have been used previously to both estimate binding affinity and reveal different conformations induced by various natural and synthetic nuclear receptor ligands (24). In addition, the assay is performed at equilibrium and bound and free ligands are not separated, so results tend to accurately reflect the relative transcriptional activation potency of the ligands in question (25). Increasing amounts of T3, the most potent form of TH ( Fig.…”

Metabolism to a response pathway selective retinoid ligand during axial pattern formation

“…Paralleling the multiplicity of retinoid receptors, several natural retinoids are known to act as retinoid receptor ligands (4,(18)(19)(20)(21)(22)(23). These fall into two groups.…”