A cDNA encoding a protein that binds retinoic acid with high affinity has been cloned. The protein is homologous to the receptors for steroid hormones, thyroid hormones and vitamin D3, and appears to be a retinoic acid-inducible trans-acting enhancer factor, suggesting that the molecular mechanisms of the effect of retinoids (vitamin A) on embryonic development, differentiation and tumour cell growth are similar to those described for other members of this nuclear receptor family.

The active derivative of vitamin A, retinoic acid (RA), is essential for normal embryonic development. The spatio-temporal distribution of embryonic RA results from regulated expression of RA-synthesizing retinaldehyde dehydrogenases and RA-metabolizing cytochrome P450s (CYP26). Excess RA administration or RA deficiency results in a complex spectrum of embryonic abnormalities. As a first step in understanding the developmental function of RA-metabolizing enzymes, we have disrupted the murine Cyp26A1 gene. We report that Cyp26A1-null mutants die during mid-late gestation and show a number of major morphogenetic defects. Spina bifida and truncation of the tail and lumbosacral region (including abnormalities of the kidneys, urogenital tract, and hindgut) are the most conspicuous defects, leading in extreme cases to a sirenomelia ("mermaid tail") phenotype. Cyp26A1 mutants also show posterior transformations of cervical vertebrae and abnormal patterning of the rostral hindbrain, which appears to be partially posteriorly transformed. These defects correlate with two major sites of Cyp26A1 expression in the rostral neural plate and embryonic tail bud. Because all of the Cyp26A1 −/− abnormalities closely resemble RA teratogenic effects, we postulate that the key function of CYP26A1 is to maintain specific embryonic areas in a RA-depleted state, to protect them against the deleterious effect of ectopic RA signaling.

We have previously described a human complementary DNA that encodes a novel protein which is homologous to members of the steroid/thyroid nuclear receptor multigene family. This novel protein (hap for hepatoma) exhibits strong homology with the human retinoic acid receptor (RAR) which has been recently characterized. To test the possibility that the hap protein might also be a retinoid receptor, a chimaeric receptor was created by replacing the putative DNA binding domain of hap with that of the human oestrogen receptor (ER). The resulting hap-ER chimaera was then tested for its ability to trans-activate an oestrogen-responsive reporter gene (vit-tk-CAT) in the presence of possible receptor ligands. Here we show that retinoic acid (RA) at physiological concentrations is effective in inducing the expression of this reporter gene by the hap-ER chimaeric receptor. This demonstrates the existence of two human retinoic acid receptors designated RAR-alpha and RAR-beta.

In addition to having profound effects on embryonic pattern formation, retinoic acid (RA) has striking effects on differentiation and maintenance of epithelial cells in vivo and in vitro Skin is a major target organ for retinoids both in its normal and pathological states. The discovery of two human nuclear receptors for RA (hRAR alpha and hRAR beta) acting as transcriptional RA-inducible enhancer factors has provided a basis for understanding how RA controls gene expression. To investigate the specific role that RARs might play during development and in adult tissues, we have cloned the mouse RAR alpha and RAR beta (mRAR alpha and mRAR beta). Their amino-acid sequences are much more homologous to those of hRAR alpha and hRAR beta, respectively, than to each other, which suggests strongly that RAR alpha- and beta-subtypes have different functions. Most interestingly we have discovered a novel RAR subtype (mRAR gamma) whose expression in adult mouse seems to be highly restricted to skin, whereas RAR alpha and RAR beta are expressed in a variety of adult tissues. Furthermore, both mRAR alpha and mRAR gamma RNAs are readily detected in undifferentiated F9 embryocarcinoma (EC) cells, whereas mRAR beta messenger RNA is induced at least 30-fold in RA-differentiated F9 cells.

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...

Cyp26b1 encodes a retinoic acid (RA) metabolizing cytochrome P450 enzyme that is expressed in embryonic tissues undergoing morphogenesis, including the testes. We have generated transgenic mice lacking Cyp26b1 and have observed increased RA levels in embryonic testes. Cyp26b1

Retinoic acid receptors (RARs) are retinoic acid (RA)-inducible enhancer factors belonging to the superfamily of steroid/thyroid nuclear receptors. We have previously characterized two human RAR (hRAR-a and hRAR-3) cDNAs and have recently cloned their murine cognates (mRAR-a and mRAR-fl) together with a third RAR (mRAR-y) whose RNA was detected predominantly in skin, a well-known target for RA. mRAR-ycDNA was used here to done its human counterpart (hRAR-y) from a T47D breast cancer cell cDNA library. Using a transient transfection assay in HeLa cells and a reporter gene harboring a synthetic RA responsive element, we demonstrate that hRAR-y cDNA indeed encodes a RAinducible transcriptional trans-activator. Interestingly, comparisons of the amino acid sequences of all six human and mouse RARs indicate that the interspecies conservation of a given member of the RAR subfamily (either a, (3, or y) is much higher than the conservation of all three receptors within a given species. These observations indicate that RAR-a, -(3, and -y may perform specific functions. We show also that hRAR-y RNA is the predominant RAR RNA species in human skin, which suggests that hRAR-y mediates some of the retinoid effects in this tissue.

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.