We have cloned a mouse cDNA homolog of P450RAI, a cytochrome P450 belonging to a new family (CYP26), which has previously been isolated from zebrafish and human cDNAs and found to encode a retinoic acid-inducible retinoic acid hydroxylase activity. The crossspecies conservation of the amino acid sequence is high, particularly between the mouse and the human enzymes, in which it is over 90%. Like its human and zibrafish counterparts, the mouse P450RAI cDNA catalyzes metabolism of retinoic acid into 4-OH-retinoic acid, 4-oxo-retinoic acid, 18-OH-retinoic acid, and unidentified water-soluble metabolites when transfected into COS-1 cells. Retinoic acid-inducible retinoic acid metabolism has previously been observed in F9 murine embryonal carcinoma cells and some derivatives lacking retinoid receptors. We were interested in determining whether P450RAI could be responsible for retinoic acid metabolism in F9 cells and in studying the effect of retinoid receptor ablation on P450RAI expression. In wild-type F9 cells and derivatives lacking RAR␥, RAR␣, and/or RXR␣, we observed a direct relationship between the level of retinoic acid metabolic activity and retinoic acid-induced P450RAI mRNA. These experiments, as well as others using synthetic receptor subtype-specific retinoids, suggest that the RAR␥ and RXR␣ receptors mediate the effects of retinoic acid on the expression of the P450RAI gene.

Two mammalian hCYP26A expression systems have been used to analyze the metabolic products of CYP26A. Through the use of extensive HPLC, UV spectroscopy, and liquid chromatography/tandem mass spectrometry (LC-MS/MS) methodology, we have conclusively demonstrated that the complex mixture of products comprises 4-OH-all-trans -retinoic acid, 4-oxo-all-trans -retinoic acid, and 18-OH-all-trans -retinoic acid, and more polar products, partially identified as dihydroxy and mono-oxo, mono-hydroxy derivatives. These more polar products are presumed to result from multiple hydroxylations on the ␤ -ionone ring. The inter-relationship of initial and polar metabolites was inferred from both gene-dose and time-course experiments. Both initial and secondary metabolic steps after 4-oxo-all-trans -retinoic acid are ketoconazole-sensitive, suggesting that steps in the production of water-soluble metabolites are cytochrome P450-dependent. -Chithalen, J. V., L. Luu, M. Petkovich, and G. Jones. HPLC-MS/MS analysis of the products generated from all-trans -retinoic acid using recombinant human CYP26A.

The retinoid X receptor (RXR) is activated by its often elusive cognate ligand, 9-cis-retinoic acid (9-cis-RA). In flies and moths, molting is mediated by a heterodimer ecdysone receptor consisting of the ecdysone monomer (EcR) and an RXR homolog, ultraspiracle (USP); the latter is believed to have diverged from its RXR origin. In the more primitive insect, Locusta migratoria (Lm), RXR is more similar to human RXRs than to USPs. LmRXR was detected in early embryos when EcR transcripts were absent, suggesting another role apart from ecdysone signaling. Recombinant LmRXRs bound 9-cis-RA and alltrans-RA with high affinity (IC 50 ‫؍‬ 61.2-107.7 nM; Kd ‫؍‬ 3 nM), similar to human RXR. To determine whether specific binding had functional significance, the presence of endogenous retinoids was assessed. Embryos were extracted by using modified Bligh and Dyer and solid-phase protocols to avoid the oily precipitate that makes this material unsuitable for assay. These extracts contained retinoids (5.4 nM) as assessed by RA-inducible Cyp26A1-promoter luciferase reporter cell lines. Furthermore, the use of HPLC and MS confirmed the presence of retinoids and identified in any embryo, 9-cis-RA, in addition to all-trans-RA. We estimate that whole embryos contain 3 nM RA, including 9-cis-RA at a concentration of 1.6 nM. These findings strongly argue for a functional role for retinoids in primitive insects and favor a model where signaling through the binding of 9-cis-RA to its RXR is established relatively early in evolution and embryonic development.all-trans-retinoic acid ͉ Locusta migratoria ͉ ultraspiracle I nsect development and metamorphosis are directed by two principal lipophilic hormones: 20-hydroxyecdysone (20-OH-Ec), the active molting hormone, and juvenile hormone (JH), whose titer determines the nature of the molt (1, 2). As demonstrated in the fruitfly, Drosophila melanogaster (Dm), 20-OH-Ec binds to the ecdysone receptor (EcR), which in turn is bound to its obligate heterodimerization partner ultraspiracle (USP), a homologue of the vertebrate retinoid X receptor (RXR) (3-6). As members of the nuclear receptor superfamily, EcR and USP/RXR share a common modular structure (7) comprised of a N-terminal variable domain (A/B), a DNA binding domain (C), hinge (D), and C-terminal ligand-binding domain (LBD; or domain E/F).The vertebrate RXRs are known heterodimeric partners of several members of the nuclear receptor superfamily, including the retinoid, thyroid, and vitamin D receptors (8). As demonstrated in vivo, these RXRs can also form homodimers and conceivably mediate an independent retinoid signaling pathway (9, 10). Indeed, the vertebrate RXRs are known ligand-activated transcription factors that bind 9-cis-retinoic acid (9-cis-RA), a stereoisomer of the vitamin A derivative, all-trans-RA (11, 12). RA receptors (RARs), reported only in vertebrates, are distinct in that they bind both all-trans-RA and 9-cis-RA with high affinity (13, 6). In contrast to the vertebrate RXRs, crystal structures reveal that DmUSP and t...