Retinoid binding-proteins redirect retinoid metabolism: biosynthesis and metabolism of retinoic acid

Many tissues and cell types, starting early in embryogenesis, convert retinol (vitamin A) into an active form, all-trans-retinoic acid. This article will discuss a current model of retinol and retinoic acid metabolism that integrates the various reactions which maintain retinoic acid homeostasis, and will also integrate the enzymology with the functions of cellular retinoid binding proteins. These conserved, high-affinity binding proteins enjoy widespread expression throughout all vertebrates and throughout most vertebrate tissues. The binding proteins limit access to retinol and retinoic acid to select enzymes and serve as substrates and affecters of retinoid metabolism.     

Diverse actions of retinoid receptors in cancer prevention and treatment

Retinoids (retinol [vitamin A] and its biologically active metabolites) are essential signaling molecules that control various developmental pathways and influence the proliferation and differentiation of a variety of cell types. The physiological actions of retinoids are mediated primarily by the retinoic acid receptors alpha, beta, and gamma (RARs) and rexinoid receptors alpha, beta, and gamma. Although mutations in RARalpha, via the PML-RARalpha fusion proteins, result in acute promyelocytic leukemia, RARs have generally not been reported to be mutated or part of fusion proteins in carcinomas. However, the retinoid signaling pathway is often compromised in carcinomas. Altered retinol metabolism, including low levels of lecithin:retinol acyl trasferase and retinaldehyde dehydrogenase 2, and higher levels of CYP26A1, has been observed in various tumors. RARbeta(2) expression is also reduced or is absent in many types of cancer. A greater understanding of the molecular mechanisms by which retinoids induce cell differentiation, and in particular stem cell differentiation, is required in order to solve the issue of retinoid resistance in tumors, and thereby to utilize RA and synthetic retinoids more effectively in combination therapies for human cancer.     

Delivery of retinoid-based therapies to target tissues

Through its various metabolites, vitamin A controls essential physiological functions. Both naturally occurring metabolites and novel retinoid analogues have shown effectiveness in many clinical settings that include skin diseases and cancer, and in animal models of human conditions affecting vision. In this review, we analyze several potential retinoid-based therapies from the point of view of drug metabolism and transport to target tissues. We focus on the endogenous factors that affect the absorption, transport, and metabolism of retinoids by taking into account data obtained from the analysis of animal models that lack the enzymes or proteins involved in the storage and absorption of retinoids. We also discuss findings of toxicity associated with retinoids in an effort to improve the outcome of retinoid-based therapies. In this context, we review evidence that esterification of retinol and retinol-based drugs within target tissues provides one of the most efficient means to improve the absorption and to reduce the toxicity associated with pharmacological doses of retinoids. Future retinoid-based therapeutic strategies could involve targeted delivery mechanisms leading to lower toxicity and improved effectiveness of retinoids.     

Distribution of endogenous retinoids,retinoid binding proteins (RBP,CRABPI) and nuclear retinoid X receptor beta (RXRbeta) in the porcine embryo

Retinoids are important signalling molecules in the development of limbs and in the determination of the anterior-posterior orientation of the embryo. The present study examined the content and distribution of retinoic acid, retinol and retinyl esters in porcine embryos during early gestation (gestation days 22-30) macroscopically and microscopically by its autofluorescence and by HPLC. Macroscopically, the yellowish-greenish autofluorescence characteristic of vitamin A was observed in tissues affected by morphogenesis, such as the limbs, in a spatial and temporal manner. Changes in the intensity of autofluorescence in the limbs paralleled changes in the concentration of retinoids in these structures. In the limbs and the body, retinol, retinyl palmitate, and all-trans-retinoic acid but neither the isomers of all-trans retinoic acid nor other retinoid metabolites were detected. In addition, the distribution of specific retinoid-binding proteins was investigated; these are involved in vitamin A transport, metabolism and signal transduction. Immunoreactive retinol-binding protein as well as cellular retinoic acid binding protein type I were only localised in the mesonephros, while the retinoid X receptor beta was widely distributed in most of the tissues and organs of the embryo throughout the time period investigated. The combination of autofluorescence and HPLC analysis allowed for the first time to attribute the yellowish-greenish autofluorescence in specific regions of the embryo to vitamin A, and offers a method to study the local cellular distribution of retinol and/or retinyl esters as well as their concentrations in embryonic tissues.     

Structure and function of cytoplasmic retinoid binding proteins

We examined the ligand protein interactions of two highly homologous cellular retinol binding proteins, CRBP and CRBP-II, and two highly homologous cellular retinoic acid binding proteins, CRABP-I and CRABP-II. While the crystal structures of all four have been determined, nuclear magnetic resonance studies provide a means for observing dynamic aspects of ligand protein interactions of these proteins in solution. The cellular functions of these proteins are less well understood. We have modeled retinoid flux between cytoplasmic retinoid proteins and model membranes and with nuclear receptors. Based on our in vitro studies, we propose that certain retinoids may indirectly influence retinoid signaling by displacing endogenous retinoids from the cytoplasmic proteins to the nuclear receptors.     

Localization of retinoid binding proteins, retinoid receptors, and retinaldehyde dehydrogenase in the chick eye

Retinoids have many functions in the eye, including, perhaps, the visual guidance of ocular growth. Therefore, we identified where retinoid receptors, binding proteins, and biosynthetic enzymes are located in the ocular tissues of the chick as a step toward discovering where retinoids are generated and where they act. Using antibodies to interphotoreceptor retinoid binding protein (IRBP), cellular retinol binding protein (CRBP), cellular retinoic acid binding protein (CRABP), cellular retinaldehyde binding protein (CRALBP), retinaldehyde dehydrogenase (RALDH), and retinoic acid receptors (RAR and RXR), we localized these proteins to cells in the retina, retinal pigmented epithelium, choroid and sclera of the chick eye. IRBP was detected in the photoreceptor layer and pigmented epithelium; CRBP was in the pigmented epithelium; CRABP was in amacrine and bipolar cells in the retina; CRALBP was in Müller cells, pigmented epithelium, choroid, and fibrous sclera; RALDH was in retinal amacrine cells, pigmented epithelium, and choroid; RAR was in amacrine cells, choroid, and chondrocytes and fibroblasts in the sclera; and RXR was in amacrine and ganglion cells, bipolar cell nuclei, choroid, and chondrocytes. We also found that the growth-modulating toxins colchicine and quisqualate destroyed selectively different subsets of CRABP-containing amacrine cells. We conclude that the distribution of proteins involved in retinoid metabolism is consistent with a role of retinoids not only in phototransduction, but also in maintenance of cellular phenotype and visual guidance of ocular growth.     

Retinyl ester formation by lecithin: retinol acyltransferase is a key regulator of retinoid homeostasis in mouse embryogenesis

The developing mammalian embryo is entirely dependent on the maternal circulation for its supply of retinoids (vitamin A and its metabolites). The mechanisms through which mammalian developing tissues maintain adequate retinoid levels in the face of suboptimal or excessive maternal dietary vitamin A intake have not been established. We investigated the role of retinyl ester formation catalyzed by lecithin:retinol acyltransferase (LRAT) in regulating retinoid homeostasis during embryogenesis. Dams lacking both LRAT and retinol-binding protein (RBP), the sole specific carrier for retinol in serum, were maintained on diets containing different amounts of vitamin A during pregnancy. We hypothesized that the lack of both proteins would make the embryo more vulnerable to changes in maternal dietary vitamin A intake. Our data demonstrate that maternal dietary vitamin A deprivation during pregnancy generates a severe retinoid-deficient phenotype of the embryo due to the severe retinoid-deficient status of the double mutant dams rather than to the lack of LRAT in the developing tissues. Moreover, in the case of excessive maternal dietary vitamin A intake, LRAT acts together with Cyp26A1, one of the enzymes that catalyze the degradation of retinoic acid, and possibly with STRA6, the recently identified cell surface receptor for retinol-RBP, in maintaining adequate levels of retinoids in embryonic and extraembryonic tissues. In contrast, the pathway of retinoic acid synthesis does not contribute significantly to regulating retinoid homeostasis during mammalian development except under conditions of severe maternal retinoid deficiency.     

Characterization of retinoid metabolism in the developing chick limb bud

Retinoids (vitamin A derivatives) have been shown to have striking effects on developing and regenerating vertebrate limbs. In the developing chick limb, retinoic acid is a candidate morphogen that may coordinate the pattern of cellular differentiation along the anteroposterior limb axis. We describe a series of investigations of the metabolic pathway of retinoids in the chick limb bud system. To study retinoid metabolism in the bud, all-trans-[3H]retinol, all-trans-[3H]retinal and all-trans-[3H]retinoic acid were released into the posterior region of the limb anlage, the area that contains the zone of polarizing activity, a tissue possibly involved in limb pattern formation. We found that the locally applied [3H]retinol is primarily converted to [3H]retinal, [3H]retinoic acid and a yet unidentified metabolite. When [3H]retinal is locally applied, it is either oxidized to [3H]retinoic acid or reduced to [3H]retinol. In contrast, local delivery of retinoic acid to the bud yields neither retinal nor retinol nor the unknown metabolite. This flow of metabolites agrees with the biochemical pathway of retinoids that has previously been elucidated in a number of other animal systems. To find out whether metabolism takes place directly in the treated limb bud, we have compared the amount of [3H]retinoid present in each of the four limb anlagen following local treatment of the right wing bud. The data suggest that retinoid metabolism takes place mostly in the treated limb bud. This local metabolism could provide a simple mechanism to generate in a controlled fashion the biologically active all-trans-retinoic acid from its abundant biosynthetic precursor retinol. In addition, local metabolism supports the hypothesis that retinoids are local chemical mediators involved in pattern formation.     

Families of retinoid dehydrogenases regulating vitamin A function: production of visual pigment and retinoic acid.

Vitamin A (retinol) and provitamin A (beta-carotene) are metabolized to specific retinoid derivatives which function in either vision or growth and development. The metabolite 11-cis-retinal functions in light absorption for vision in chordate and nonchordate animals, whereas all-trans-retinoic acid and 9-cis-retinoic acid function as ligands for nuclear retinoic acid receptors that regulate gene expression only in chordate animals. Investigation of retinoid metabolic pathways has resulted in the identification of numerous retinoid dehydrogenases that potentially contribute to metabolism of various retinoid isomers to produce active forms. These enzymes fall into three major families. Dehydrogenases catalyzing the reversible oxidation/reduction of retinol and retinal are members of either the alcohol dehydrogenase (ADH) or short-chain dehydrogenase/reductase (SDR) enzyme families, whereas dehydrogenases catalyzing the oxidation of retinal to retinoic acid are members of the aldehyde dehydrogenase (ALDH) family. Compilation of the known retinoid dehydrogenases indicates the existence of 17 nonorthologous forms: five ADHs, eight SDRs, and four ALDHs, eight of which are conserved in both mouse and human. Genetic studies indicate in vivo roles for two ADHs (ADH1 and ADH4), one SDR (RDH5), and two ALDHs (ALDH1 and RALDH2) all of which are conserved between humans and rodents. For several SDRs (RoDH1, RoDH4, CRAD1, and CRAD2) androgens rather than retinoids are the predominant substrates suggesting a function in androgen metabolism as well as retinoid metabolism.     

Metabolism and transactivation activity of 13,14-dihydroretinoic acid

The metabolism of vitamin A is a highly regulated process that generates essential mediators involved in the development, cellular differentiation, immunity, and vision of vertebrates. Retinol saturase converts all-trans-retinol to all-trans-13,14-dihydroretinol (Moise, A. R., Kuksa, V., Imanishi, Y., and Palczewski, K. (2004) J. Biol. Chem. 279, 50230-50242). Here we demonstrate that the enzymes involved in oxidation of retinol to retinoic acid and then to oxidized retinoic acid metabolites are also involved in the synthesis and oxidation of all-trans-13,14-dihydroretinoic acid. All-trans-13,14-dihydroretinoic acid can activate retinoic acid receptor/retinoid X receptor heterodimers but not retinoid X receptor homodimers in reporter cell assays. All-trans-13,14-dihydroretinoic acid was detected in vivo in Lrat-/- mice supplemented with retinyl palmitate. Thus, all-trans-13,14-dihydroretinoic acid is a naturally occurring retinoid and a potential ligand for nuclear receptors. This new metabolite can also be an intermediate in a retinol degradation pathway or it can serve as a precursor for the synthesis of bioactive 13,14-dihydroretinoid metabolites.     

Effects of retinoid beta-glucuronides and N-retinoyl amines on the differentiation of HL-60 cells in vitro

Retinoyl beta-glucuronide and retinyl beta-glucuronide, which are naturally occurring water-soluble metabolites of vitamin A, induce the granulocytic differentiation of HL-60 cells in vitro, as evidenced by an increased reduction of nitroblue tetrazolium. The relative effectiveness of various retinoids in differentiation is retinoic acid greater than retinoyl beta-glucuronide greater than retinyl beta-glucuronide. Under the selected assay conditions, retinol, hydroxyphenyl-retinamide, retinamide, and N-retinoyl-phenylalanine are essentially inactive in differentiation. At concentrations of retinoids from 10(-9) to 10(-5) M, cell viability was best with the retinoid beta-glucuronides and retinamide, less with retinoic acid and retinol, and poorest with the N-retinoyl aromatic amines. Cellular growth was depressed only slightly by retinyl beta-glucuronide and retinamide, but to a greater degree by the other derivatives. Retinoyl beta-glucuronide was hydrolyzed in part to retinoic acid, whereas retinyl beta-glucuronide was cleaved to retinol, if at all, at a very slow rate. Under the selected assay conditions, retinoic acid and the retinoid beta-glucuronides primarily induce the differentiation of HL-60 cells, whereas the N-retinoyl aromatic amines show cytotoxicity.     

Altered retinoid distribution in the repeated epilation (Er) mutant mouse.

The repeated epilation (Er) mutation in mice causes successive loss and regrowth of hair in heterozygotes (Er/+), and blocks orofacial development and epidermal differentiation in lethal homozygotes (Er/Er). Because the mutation affects a systemic factor, because the Er phenotypes resemble exposure to excess retinoids and because retinoids are critical regulators of epithelial differentiation, we examined whether systemic retinoid levels are altered by the Er defect. Liver retinoic acid and retinol were elevated 1.5- and 3.5-fold, respectively, in adult heterozygotes (Er/+) compared to normal (+/+) animals. Retinyl palmitate was increased 2-fold in heterozygous skin and 3-fold in kidney, but the retinol level in plasma was only half that of normal animals. Newborn heterozygous liver also had nearly 2-fold increased retinoids compared to normal. In contrast, Er/Er newborns had reduced retinoid levels in liver, two-thirds the retinol and 15% the retinyl palmitate compared to normal, but greater than 4-fold elevated levels of retinyl palmitate in the extrahepatic body. Tissue contents of retinol binding protein (RBP), which transports vitamin A from the liver to the remainder of the body, were determined by immunoblotting with anti-mouse RBP. Newborn normal and mutant animals had similar liver microsomal RBP contents. RBP contents in plasma and in liver microsomes were also similar in normal and Er/+ adults despite different retinol contents in the Er/+ tissues. Hair follicles of the Er/+, but not the normal adult, were stained with this antiserum to RPB in the outer root sheath layer. These results strongly suggest that altered retinoid distribution is associated with, and may be responsible for, the altered epithelial differentiation in the Er mutant.     

A novel retinoid binding property of human annexin A6

Vitamin A (all-trans retinol) and all-trans retinoid acid (ATRA) interacted with human annexin A6 (AnxA6) as evidenced by AnxA6-induced blue shift of retinoid absorption maxima, by AnxA6-Trp fluorescence quenching and by a fluorescence resonance energy transfer from a Trp residue of AnxA6 to retinol. In addition, both retinoids stimulated the calcium-dependent binding of AnxA6 to liposomes, accompanied by oligomerization of AnxA6. Up to our knowledge, it is a first report supporting the hypothesis of a direct implication of AnxA6 in vitamin A-dependent tissue mineralization.     

Enzymes and binding proteins affecting retinoic acid concentrations

Free retinoids suffer promiscuous metabolism in vitro. Diverse enzymes are expressed in several subcellular fractions that are capable of converting free retinol (retinol not sequestered with specific binding proteins) into retinal or retinoic acid. If this were to occur in vivo, regulating the temporal-spatial concentrations of functionally-active retinoids, such as RA (retinoic acid), would be enigmatic. In vivo, however, retinoids occur bound to high-affinity, high-specificity binding proteins, including cellular retinol-binding protein, type I (CRBP) and cellular retinoic acid-binding protein, type I (CRABP). These binding proteins, members of the superfamily of lipid binding proteins, are expressed in concentrations that exceed those of their ligands. Considerable data favor a model pathway of RA biosynthesis and metabolism consisting of enzymes that recognize CRBP (apo and holo) and holo-CRABP as substrates and/or affecters of activity. This would restrict retinoid access to enzymes that recognize the appropriate binding protein, imparting specificity to RA homeostasis; preventing, e.g. opportunistic RA synthesis by alcohol dehydrogenases with broad substrate tolerances. An NADP-dependent microsomal retinol dehydrogenase (RDH) catalyzes the first reaction in this pathway. RDH recognizes CRBP as substrate by the dual criteria of enzyme kinetics and chemical crosslinking. A cDNA of RDH has been cloned, expressed and characterized as a short-chain alchol dehydrogenase. Retinal generated in microsomes from holo-CRBP by RDH supports cytosolic RA synthesis by an NAD-dependent retinal dehydrogenase (RalDH). RalDH has been purified, characterized with respect to substrate specificity, and its cDNA has been cloned. CRABP is also important to modulating the steady-state concentrations of RA, through sequestering RA and facilitating its metabolism, because the complex CRABP/RA acts as a low K_m substrate.     

Retinoid status and responsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking retinoid binding protein or retinoid receptor forms

We have investigated the role of Vitamin A (retinoid) proteins in hepatic retinoid processing under normal conditions and during chemical stress induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a chemical known to interfere with retinoid turnover and metabolism. Three separate studies were performed in wildtype control mice and transgenic mice that lack one or more isoforms of retinoic acid receptors (RAR), retinoid X receptors (RXR), or intracellular retinoid-binding proteins (CRABP I, CRABP II, CRBP I). Body and organ weight development was monitored from 2 weeks of age to adult, and hepatic levels of retinyl esters, retinol, and retinoic acid were investigated. In addition, hepatic concentrations of 9-cis-4-oxo-13,14-dihydro-retinoic acid, a recently discovered retinoid metabolite that has proven sensitive to both TCDD exposure and Vitamin A status, were also determined. Mice absent in the three proteins CRBP I, CRABP I, and CRABP II (CI/CAI/CAII-/-) displayed significantly lower hepatic retinyl ester, retinol, and all-trans-retinoic acid levels compared to wildtype mice, whereas the liver concentrations of 9-cis-4-oxo-13,14-dihydro-retinoic acid was considerably higher. After treatment with TCDD, hepatic total retinoids were almost entirely depleted in the CI/CAI/CAII-/- mice, whereas wildtype mice and mice lacking CRABP I, and CRABP II (CAI/CAII-/-) retained approximately 60-70% of their Vitamin A content compared to controls at 28 days. RAR and RXR knockout mice responded similarly to wildtype mice with respect to TCDD-induced retinoid disruption, with the exception of RXRbeta-/- mice which showed no decrease in hepatic Vitamin A concentration, suggesting that the role of RXRbeta in TCDD-induced retinoid disruption should be further investigated. Overall, the abnormal retinoid profile in the triple knockout mice (CI/CAI/CAII-/-), but not double knockout (CAI/CAII-/-) mice, suggests that a loss of CRBP I may account for the difference in retinoid profile in CI/CAI/CAII-/- mice, and is likely to result in an increased susceptibility to hepatic retinoid depletion following dioxin exposure.     

Chylomicron remnant-vitamin A metabolism by the human hepatoma cell line HepG2

The binding and metabolism of [3H]vitamin A-containing chylomicron (CM) remnants by the human hepatoma cell line HepG2 were studied. Mesenteric lymph chylomicrons were collected from [3H]retinol-fed rats and incubated with lipoprotein lipase to obtain CM remnants. At 4 degrees C, specific CM remnant binding was inhibited by an excess of unlabeled CM remnants. Specific binding predominated at low concentrations and approached saturation while total binding continued to increase over an extensive concentration range (0.45-32 microgram triglyceride/ml). CM remnant uptake at 37 degrees C was greater than that of CM and at least 70 times more efficient than the pinocytosis of sucrose. CM remnant binding increased with the extent of lipolysis. Addition of human apolipoprotein E enhanced both CM remnant and CM binding. After internalization, HepG2 cells hydrolyzed CM remnant-[3H]retinyl esters, and radiolabeled metabolites accumulated. As a function of the concentration of [3H]retinoid initially bound to cells, retinol and retinyl esters accumulated as the major cell-associated metabolites. In contrast, retinol was the major metabolite in the medium only at low retinoid concentrations; other more polar metabolites accumulated at higher concentrations (greater than 110 pmol retinoid/mg cell protein). The accumulation in the medium of labeled metabolites derived from CM remnant-retinoid was reduced when cells were preincubated in unlabeled retinol-supplemented media. The specific activity of retinol in the medium indicated that CM remnant-vitamin A had mixed with the cellular store prior to its secretion as retinol. These results indicate that HepG2 cells internalize CM remnants in part by specific binding sites, and that the metabolism of CM remnant-retinoids by the HepG2 cell involves retinyl ester hydrolysis and the secretion of retinol and other more polar metabolites. These processes were regulated in part by the concentration of retinoid delivered by the CM remnant and by the initial retinoid content of the cell.