Evidence suggests that 4-hydroxylation of RA inside the target cell limits its biological activity and initiates a degradative process of RA leading to its eventual elimination. However, 18-hydroxylation and glucuronidation may also be important steps in this process. In this paper, we describe the cloning and characterization of the first mammalian retinoic acid-inducible retinoic acid-metabolizing cytochrome P450 (hP450RAI), which belongs to a novel class of cytochromes (CYP26). We demonstrate that hP450RAI is responsible for generation of several hydroxylated forms of RA, including 4-OH-RA, 4-oxo-RA, and 18-OH-RA. We also show that hP450RAI mRNA expression is highly induced by RA in certain human tumor cell lines and further show that RA-inducible RA metabolism may correlate with P450RAI expression. We conclude that this enzyme plays a key role in RA metabolism, functioning in a feedback loop where RA levels are controlled in an autoregulatory manner.Regulation of retinoid signaling may be controlled by a number of coordinated mechanisms, including retinoid synthesis, cell-specific expression of retinoid-binding proteins and nuclear receptors, and metabolism of retinoids (for review see Refs. 1-3). The generation of RA 1 from its precursors, retinol and retinaldehyde, and its catabolism to more polar hydroxylated forms such as 4-OH-RA, 4-oxo-RA, and 18-OH-RA are counterbalanced metabolic pathways that regulate RA levels in RAsensitive tissues (4, 5). Cellular retinoic acid-binding proteins may also play a role in establishing this balance by sequestering high levels of RA (6). There is considerable evidence to suggest that 4-OH-, 4-oxo-, and 18-OH-RA are polar intermediates in the catabolism and eventual elimination of RA (5,7,8). Thus both sequestration and metabolism may function to protect RA-sensitive tissues from deleterious concentrations of RA.We have cloned and characterized cDNAs corresponding to a retinoic acid-inducible gene encoding a human cytochrome P450-related hydroxylase (P450RAI) responsible for generation of multiple hydroxylated products of RA. hP450RAI appears to be the human ortholog of the previously characterized zebrafish P450RAI (zP450RAI) (9), indicating that this important cytochrome is highly conserved structurally and functionally across species. We also demonstrate that hP450RAI is inducible by RA in a number of different cell types. We speculate that this enzyme plays a key role in determining the metabolic fate of endogenous retinoids and may also be implicated in the clearance of exogenous retinoids administered therapeutically. MATERIALS AND METHODScDNA Library Screening-A NTERA2-D1 cDNA library (Stratagene) was screened according to the manufacturer's directions. Briefly, 1.0 ϫ 10 Ϫ6 independent plaques were screened using a random-primed, ␣-[ 32 P]dATP-labeled full-length zP450RAI cDNA. Filters were prehybridized for 4 h at 37°C in 50% formamide, 5 ϫ SSPE, 1 ϫ Denhardt's (without bovine serum albumin), 0.2 mg/ml denatured salmon sperm DNA. Hybridization was performed overn...

Retinoic acid (RA) metabolites of vitamin A are key regulators of gene expression involved in embryonic development and maintenance of epithelial tissues. The cellular effects of RA are dependent upon the complement of nuclear receptors expressed (RARs and RXRs), which transduce retinoid signals into transcriptional regulation, the presence of cellular retinoid-binding proteins (CRABP and CRBP), which may be involved in RA metabolism, and the activity of RA metabolizing enzymes. We have been using the zebrafish as a model to study these processes. To identify genes regulated by RA during exogenous RA exposure, we utilized mRNA differential display. We describe the isolation and characterization of a cDNA, P450RAI, encoding a novel member of the cytochrome P450 family. mRNA transcripts for P450RAI are expressed normally during gastrulation, and in a defined pattern in epithelial cells of the regenerating caudal fin in response to exogenous RA. In COS-1 cells transfected with the P450RAI cDNA, alltrans-RA is rapidly metabolized to more polar metabolites. We have identified 4-oxo-RA and 4-OH-RA as major metabolic products of this enzyme. P450RAI represents the first enzymatic component of RA metabolism to be isolated and characterized at the molecular level and provides key insight into regulation of retinoid homeostasis.

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