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.
A full-length cDNA for the human liver mitochondrial cytochrome P-450 CYP27 was cloned from a human hepatoma HepG2 cDNA library and then subcloned into the mammalian expression vector pSG5. When CYP27 cDNA was trnsfected into COS-1 tansformed monkey kidney ceRs along with adrenodoin cDNA, transected cells revealed a 10-to 20-fold higher vitamin D3-25-hydroxylase activity than nontransfected cells. Transfected cells were capable of 25-hydroxylation of vitamin D3, la-hydroxyvitamin D3 and lahydroxydihydrotachysterol3. In each case they also showed the ability to 26(27)-hydroxylate the cholesterol-like (D3) side chain. The relative rates of 25-and 26(27)-hydroxylation of la-hydroxyvitamin D3 approximately mimicked the ratio of products observed in HepG2 cells. Vitamin D2 and lahydroxyvitamin D2, both with the ergosterol-like side chain, were 24-and 26(27)-hydroxylated by CYP27. The rate of side-chain 24-, 25-, or 26(27)-hydroxylation was greater for la-hydroxylated vitamin D analogs than for their nonhydroxylated counterparts. We conclude that CYP27 is capable of24-, 25-, and 26(27)-hydroxylation of vitamin D analogs and that the nature of products Is partially dictated by the side chain of the substrate. This work has revealed that the cytochrome P-450 CYP27 may be important in the metabolism of vitamin D analogs used as drugs.The cytochrome P45S-containing enzyme vitamin D3 25-hydroxylase has been extensively studied over the past 20 years or so since the first broken-cell systems were described (1). Subsequently, many studies involving purification and characterization ofthe enzyme(s) have been performed using the liver of different experimental animals, in particular that of the rat and rabbit (2, 3). In the rat, both microsomal and mitochondrial versions of the vitamin D3 25-hydroxylase have been reported (3, 4). The human liver counterpart was believed to exist exclusively in the mitochondrial inner membrane (5). This conclusion was subsequently challenged by the observation that the microsomal fraction from human liver might contain an inhibitor(s) of the enzyme (6).The cloning of the liver mitochondrial vitamin D3 25-hydroxylase cDNA has been reported for rat (7) and rabbit (8, 9). The cloning of the presumed human homolog of the liver mitochondrial vitamin D3 25-hydroxylase has also been reported (10, 11) and this cytochrome P-450 has been named CYP27. Though Cali and coworkers (10,12) (Kingston, ON).Cell Culture and Incubation with Vitamin D Analogs. HepG2 cells were cultured as described (14). COS-1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (penicillin G, 100 pg/ml; gentamicin, 5 pg/ml; and Fungizone, 300 ng/ml).Tissue culture plates (100-or 150-mm diameter) were washed twice with phosphate-buffered saline and once with medium containing antibiotics plus 1% (wt/vol) bovine serum albumin. Serum-free medium containing 1% albumin (4 ml per 100-mm plate; 10 ml per 150-mm plate) was added to each plate, followed by vitamin D anal...
Human CYP27A1 is a mitochondrial cytochrome P450, which is principally found in the liver and plays important roles in the biological activation of vitamin D(3) and in the biosynthesis of bile acids. We have applied a systematic analysis of hydrogen bonding patterns in 11 prokaryotic and mammalian CYP crystal structures to construct a homology-based model of CYP27A1. Docking of vitamin D(3) structures into the active site of this model identified potential substrate contact residues in the F-helix, the beta-3 sheet, and the beta-5 sheet. Site-directed mutagenesis and expression in COS-1 cells confirmed that these positions affect enzymatic activity, in some cases shifting metabolism of 1alpha-hydroxyvitamin D(3) to favor 25- or 27-hydroxylation. The results suggest that conserved hydrophobic residues in the beta-5 hairpin help define the shape of the substrate binding cavity and that this structure interacts with Phe-248 in the F-helix. Mutations directed toward the beta-3a strand suggested a possible heme-binding interaction centered on Asn-403 and a structural role for substrate contact residues Thr-402 and Ser-404.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.