Immunohistochemical localization of retinoid binding proteins at the materno-fetal interface of the porcine epitheliochorial placenta

Retinol and retinoic acid that are potent modulators of gene expression are vital for development and growth of the conceptus. Apart from being transported across the placenta, retinol and retinoic acid may also be active in the placenta per se. Three proteins involved in 1) serum transport of retinol (retinol binding protein [RBP]), 2) cellular transport and metabolism of retinol (cellular RBP [CRBP] I), and 3) retinoic acid (cellular retinoic acid binding protein [CRABP] I), respectively, have been located by immunohistochemistry during gestation in the porcine placenta. This is a diffuse epitheliochorial placenta composed of areolar-gland subunits, where transport of larger molecules takes place, and interareolar regions, where gas-exchange and trophoblast absorption of hemotroph occur. Immunoreactive-RBP (ir-RBP) as well as CRBP I (ir-CRBP) was detected in uterine glands and in areolar trophoblasts, suggesting that RBP-retinol is secreted by the glands and absorbed by the trophoblasts. Both proteins were present also at the interareolar regions, with ir-CRBP in both the uterine epithelium and the apposing trophoblasts, but ir-RBP only in the former. The localization of ir-CRABP was, in contrast, strictly limited to interareolar trophoblasts. Together these findings suggest that 1) the areolar gland subunits are important for transport of retinol and retinol-RBP, and 2) retinoid binding proteins are involved in the development and growth of the porcine placenta.     

细胞内视黄酸信号传递系统

视黄酸对基因表达的调控与肿瘤细胞的分化、胚胎的发育以及疾病的发生关系密切．视黄酸的基因调控作用是通过视黄酸信号传递系统实现的．视黄酸信号传递系统包括视黄酸、细胞液视黄醇（酸）结合蛋白、视黄酸细胞核受体及视黄酸反应元件等．视黄酸信号传递系统自成一体系,在这一系列调控的级联反应中存在着多级反馈调控环节,而且该系统还与视黄酸配体以外的信号系统相联系．     

Mouse conceptuses have a limited capacity to elevate the mRNA level of cellular retinoid binding proteins in response to teratogenic doses of retinoic acid.

In these studies, we wished to determine the effect of teratogenic doses of retinoic acid on the expression of cellular retinoic acid binding protein I (CRABP-I) mRNA, cellular retinoic acid binding protein II (CRABP-II) mRNA, cellular retinol binding protein I (CRBP-I) mRNA, and cellular retinol binding protein II (CRBP-II) mRNA in mouse conceptuses. Levels of CRABP-II mRNA and CRBP-I mRNA were modestly elevated (2.5-fold and 1.5-fold, respectively) in 9-day gestation conceptuses following treatment of dams with 100 mg/kg b.w. of retinoic acid. These levels were elevated by 6 hr following treatment and remained elevated until 48 and 24 hr, respectively. Two other retinoids, etretinate and retinoyl beta-glucuronide, also moderately elevated CRABP-II mRNA and CRBP-I mRNA levels in conceptuses. In contrast, the levels of CRABP-I mRNA in the conceptuses remained unaffected by treatment with any of these three retinoids. These results demonstrate that conceptuses have a limited capacity to elevate the cellular retinoid binding proteins mRNA levels and presumably the synthesis of their respective proteins in response to high, teratogenic doses of retinoic acid. As a result, an excess of free retinoic acid becomes available to the nuclear retinoic acid receptors, which may lead to inappropriate gene expression and eventual maldevelopment.     

Transfer of retinoic acid from its complex with cellular retinoic acid-binding protein to the nucleus

Cellular retinoic acid-binding protein (CRABP), a potential mediator of retinoic acid action, enables retinoic acid to bind in a specific manner to nuclei and chromatin isolated from testes of control and vitamin A-deficient rats. The binding of retinoic acid was followed after complexing [3H]retinoic acid with CRABP purified from rat testes. The binding was specific, saturable, and temperature dependent. If CRABP charged with nonlabeled retinoic acid was included in the incubation, binding of radioactivity was diminished, whereas inclusion of free retinoic acid, or the complex of retinol with cellular retinol binding protein (CRBP) or serum retinol binding protein had no effect. Approximately 4.0 X 10(4) specific binding sites for retinoic acid were detected per nucleus from deficient animals. The number of binding sites observed was influenced by vitamin A status. Refeeding vitamin A-deficient rats (4 h) with retinoic acid lowered the amount of detectable binding sites in the nucleus. CRABP itself did not remain bound to these sites, indicating a transfer of retinoic acid from its complex with CRABP to the nuclear sites. Further, CRBP, the putative mediator of retinol action, was found to enable retinol to be bound to testicular nuclei, in an interaction similar to the binding of retinol to liver nuclei described previously.     

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.     

Functional studies of newly synthesized benzoic acid derivatives: identification of highly potent retinoid-like activity

Three newly synthesized benzoic acid derivatives (terephthalic acid anilides, chalcone carboxylic acid, and azobenzene carboxylic acid), with a certain structural similarity to retinoic acid, were examined for their retinoid-like bioactivity and their capacity to bind to cellular retinoid binding proteins. Two in vitro systems were used to evaluate their retinoid-like bioactivity: inhibition of adipose conversion of ST 13 murine preadipose cells and growth promotion of murine sarcoma virus (MSV)-transformed 3T3 cells in serum-free culture. All three compounds tested inhibited ST 13 adipose conversion at nanomolar concentrations in a manner similar to classical retinoids such as retinoic acid. The growth-stimulating activity of these compounds on MSV-transformed 3T3 cells was one to two orders of magnitude greater than that of retinoic acid. Simultaneous treatment with these compounds and retinoic acid produced only a barely detectable additive effect, suggesting a common mechanism of action, whereas unrelated mitogens, thrombin, and insulin worked synergistically in combination with retinoic acid. None of the compounds competed with retinol for binding to cellular retinol binding protein. However, two of the three competed with retinoic acid for binding to cellular retinoic acid binding protein. This study provides evidence that the newly synthesized compounds should be included among the retinoids and that their strong biological activity will undoubtedly contribute to the biological and medical application of retinoids.     

Retinoic acid-binding protein in rat tissue. Partial purification and comparison to rat tissue retinol-binding protein.

When the 100,000 X g supernatant fractions of several rat organs are incubated with all-trans-[3H]retinoic acid, a binding component for retinoic acid with a sedimentation coefficient of 2 S can be detected by sucrose gradient centrifugation. This tissue binding protein for retinoic acid is distinct from the tissue binding protein for retinol which has been previously described. The tissue retinoic acid-binding protein has been partially purified from rat testis and this partially purified protein would appear to have a molecular weight of 14,500 as determined by gel filtration and high binding specificity for all-trans-retinoic acid. Binding of [3H]retinoic acid is not diminished by a 200-fold molar excess of retinal, retinol, or oleic acid but is reduced by a 200-fold excess of unlabeled retinoic acid. Tissue retinoic acid-binding protein can be detected in extracts of brain, eye, ovary, testis, and uterus but is apparently absent in heart muscle, small intestine, kidney, liver, lung, gastrocnemious muscle, serum, and spleen. This distribution is different than that observed for the tissue retinol-binding protein. Tissue retinol-binding protein was also purified extensively from rat testis. The partially purified protein has an apparent molecular weight of 14,000 and high binding specificity for all-trans-[3H]retinol as only unlabeled all-trans-retinol but not retinal, retinoic acid, retinyl acetate, retinyl palmitate, or oleic acid could diminish binding of the 3H ligand under the conditions employed. The partially purified protein has a fluorescence excitation spectrum with lambda max at 350 nm. In contrast, the retinol-binding protein isolated from rat serum and described by others has a fluorescence excitation spectrum with lambda max at 334 nm and an apparent molecular weight of 19,000. When partially purified tissue retinol-binding protein is extracted with heptane, the heptane extract has a fluorescence excitation spectrum similar to that of all-trans-retinol.     

Novel retinoid-binding proteins from filarial parasites.

The present study deals with the discovery and partial characterization of specific binding proteins for retinol and retinoic acid from filarial parasites (worms of the superfamily Filarioidea), including those from two species of Onchocerca. These binding proteins, which are distinct in their physicochemical properties and in the mode of ligand interactions from the host-tissue retinoid-binding proteins, may be involved in the mediation of the putative biological roles of retinoids in the control of parasitic growth, differentiation and reproduction. Parasite retinol-binding protein and retinoic acid-binding protein exhibited specificity for binding retinol and retinoic acid respectively. Both the binding proteins showed an s20,w value of 2.0 S. On gel filtration, both proteins were retarded to a position corresponding to the same molecular size (19.0 kDa). On preparative columns, the parasite binding proteins exhibited isoelectric points at pH 5.7 and 5.75. Unlike the retinoid-binding proteins of mammalian and avian origin, the parasite retinoid-binding proteins showed a lack of mercurial sensitivity in ligand binding. The comparative amounts of retinoic acid-binding protein in five parasites, Onchocerca volvulus, Onchocerca gibsoni, Dipetalonema viteae, Brugia pahangi and Dirofilaria immitis, were between 2.7 and 3.1 pmol of retinoic acid bound/mg of extractable protein. However, the levels of parasite retinol-binding protein were between 4.8 and 5.8 pmol/mg, which is considerably higher than the corresponding levels of cellular retinol-binding protein of mammalian and avian origin. Both retinol- and retinoic acid-binding-protein levels in O. volvulus-infected human nodules and O. gibsoni-infected bovine nodules were similar to their levels in mammalian tissues. Also, these nodular binding proteins, like the host-binding proteins, exhibited mercurial sensitivity to ligand interactions.     

Ovomucoid and ovoinhibitor isolated from chicken egg white are immunologically cross-reactive

We report the first application of high pressure liquid chromatography (HPLC) in the rapid detection of cellular retinoic acid binding protein (CRABP) and cellular retinol binding protein (CRBP). Cytosols from cultured cells (3T6 and MCF-7) or from tumors (melanoma and ovarian) were labeled with [3H]retinoic acid (30 Ci/mmol) and [3H]retinol (43 Ci/mmol) and analyzed via HPLC employing a 60 cm TSK 3000 sw column. In each case CRABP and CRBP were readily detectable at an elution volume of 22.5 ml, consistent with their molecular weights of 14,600. Identity of the binding protein peaks was established by saturability, specificity, and selective inhibition of binding by an organomercurial. Thus, this method, which resolves CRABP and CRBP in crude mixtures from the majority of cytosolic proteins, should be a valuable tool in the evaluation of vitamin A-binding protein interactions and their biological significance.     

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.     

Separable binding proteins for retinoic acid and retinol in bovine retina.

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [3H] retinoic acid which could not be effectively displaced by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [3H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.     

The biosynthesis of retinoic acid from retinol by rat tissues in vitro

This report shows that a spectrum of vitamin A-dependent tissues can produce retinoic acid by synthesis in situ, indicates that cellular retinol and retinoic acid binding proteins are not obligatory to retinoic acid synthesis, and provides initial characterization of retinoic acid synthesis by rat tissues. Retinoic acid synthesis from retinol was detected in homogenates of rat testes, liver, lung, kidney, and small intestinal mucosa, but not spleen. Zinc did not stimulate the conversion of retinol into retinoic acid by liver homogenates. Retinoic acid synthesis was localized in cytosol of liver and kidney, where its rate of synthesis from retinol was fourfold (liver) and sevenfold (kidney) slower than from retinal. The synthesis of retinoic acid from retinol required NAD and was not supported by NADP. NADH (0.5 mM) reduced retinoic acid synthesis from retinol, supported by NAD (2 mM), by 50-70%, but was fivefold less potent in reducing retinoic acid synthesis from retinal. Dithiothreitol enhanced the conversion of retinol, but not retinal, into retinoic acid. EDTA inhibited the conversion of retinol into retinoic acid slightly (13%, liver; 29%, kidney). A high ethanol concentration (100 mM), relative to retinoid substrate (10 microM), inhibited retinoic acid synthesis from retinol (liver, 54%; kidney, 30%) and from retinal (30%, liver; 9%, kidney). 4'-(9-Acridinylamino)methansulfon-m-anisidine, an inhibitor of aldehyde oxidase, and disulfiram, a sulfhydryl-group crosslinking agent, were potent inhibitors of retinoic acid synthesis at 10 microM or less, and seemed equipotent in liver and kidney. 4-Methylpyrazole, an inhibitor of ethanol metabolism, also inhibited retinoic acid synthesis from retinol, but was less potent than the former two inhibitors, and affected liver to a greater extent than kidney, particularly with retinal as substrate.     

Rapid characterization of cellular retinoid-binding proteins by high-performance size-exclusion chromatography

A high-performance size-exclusion chromatographic method was developed for the analysis of cellular retinol and retinoic acid-binding proteins. The chromatographic analysis of the retinol-retinol-binding protein complex or the retinoic acid-retinoic acid-binding protein complex requires 15 min. The use of high-specific-radioactivity retinoid ligand (30-40 Ci/mmol) allows routine detection of 25 fmol of retinoid-binding protein/mg of cytosolic protein. Thus, this method provides a rapid alternative to sucrose density gradient sedimentation analysis of the cellular retinoid-binding proteins. High-performance size-exclusion chromatography is well suited to screening novel tissues, tumors, and cell lines for the presence of retinoid-binding proteins and to the further characterization of these cellular proteins. This method was applied to the characterization of cellular retinol- and retinoic acid-binding proteins in fetal rabbit tissues. Both retinol- and retinoic acid-binding proteins were detected in fetal rabbit brain, intestine, kidney, and lung at a gestational age of 28 days. Neither retinoid-binding protein was detected in 28-day-old fetal rabbit placenta. Specific retinoic acid binding in fetal intestinal cytosol decreased as a function of increasing gestational age.     

Immunolocalization of retinoic acid biosynthesis systems in selected sites in rat

Vitamin A deficiency leads to focal metaplasia of numerous epithelial tissues with altered differentiation from columnar (in general) to stratified squamous cells. This process can be reversed with vitamin A repletion. Previously, we described a system of retinoic acid (RA) synthesis in the cycling rat uterus consisting of cellular retinol binding protein (Crbp), epithelial retinol dehydrogenase (eRoldh), retinal dehydrogenase 2 (Aldh1a2), and cellular retinoic acid binding protein type II (Crabp2). Western blot analysis, RT-PCR, and immunohistochemistry were performed to test whether this retinoic acid synthesis system was also present in other vitamin A sensitive tissues. We found that combinations of Crbp, eRoldh, Aldh1a2 or Aldh1a3, and Crabp2 were present in all vitamin A sensitive tissues examined. In the ureter, while eRoldh was present, another short chain alcohol dehydrogenase reductase (possibly Roldh 1, 2, or 3) was in higher concentration in the transitional epithelia. In several tissues, Crbp, Aldh1a2, and/or Aldh1a3 localized to mesenchyme and/or epithelial cells, while eRoldh and Crabp2 were expressed only in epithelial cells. This suggests that mesenchymal-epithelial interactions may be as important in the adult as they are during development and that local synthesis of RA is important in maintenance of these tissues.     

A close structural relationship of rat liver Z-protein to cellular retinoid binding proteins and peripheral nerve myelin P2 protein

Striking sequence homologies were found among the major cytosolic lipid and dye binding protein (Z-protein), cellular retinoid binding proteins and peripheral nerve myelin P2 protein. In addition, the presence of a number of cytosolic lipid binding (or exchange) proteins having similar molecular weights and amino acid compositions suggests a new family of intracellular proteins with high affinities to lipids, possibly diverged from a common ancestor. In view of regulatory roles of these proteins in lipid metabolism and transport, myelin P2 protein may also possess a similar physiological function(s).     

Ascaridia galli fatty acid-binding protein, a member of the nematode polyprotein allergens family.

A fatty acid-binding protein from the nematode Ascaridia galli was characterized. The gene was isolated and recombinantly expressed in Escherichia coli. According to the deduced amino acid sequence A. galli fatty acid-binding protein (AgFABP) belongs to the family of nematode polyprotein allergens, as shown by Western blotting and PCR analysis with genomic DNA and cDNA. Both native and recombinant proteins bind fatty acids and retinoids with high affinity. The fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1-sulfonyl)amino] undecanoic acid (DAUDA) shows substantial changes in its emission spectrum when bound to AgFABP; this binding is reversed by fatty acids such as oleate. Moreover, changes of the intrinsic fluorescence of retinol and retinoic acid confirm retinoid binding activity of AgFABP. Fluorescence titration experiments with DAUDA indicate stoichiometric binding to a single binding site per monomer unit with affinities (Kd) of 1.6 and 1.8 x 10(-7) m for native and the recombinant protein, respectively. The apparent binding affinities of the nonfluorescent ligands were calculated in displacement experiments with DAUDA and values in the same range were obtained for myristic, palmitic, oleic, linoleic, arachidonic and retinoic acid. Additionally, the binding affinity of AgFABP for oleate and palmitate was determined by direct and indirect radiochemical analysis and the values obtained were similar to those from the fluorescent experiments. Both proteins show a preference for the binding of long-chain saturated and unsaturated fatty acids, but not for short chain (C3-C12) and branched fatty acids, cholesterol and tryptophan.     

Separable binding proteins for retinoic acid and retinol in bovine retina

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [³H] retinoic acid which could not be effectively displayed by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [³H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.     

Folding of intracellular retinol and retinoic acid binding proteins

The folding mechanisms of cellular retinol binding protein II (CRBP II), cellular retinoic acid binding protein I (CRABP I), and cellular retinoic acid binding protein II (CRABP II) were examined. These beta-sheet proteins have very similar structures and higher sequence homologies than most proteins in this diverse family. They have similar stabilities and show completely reversible folding at equilibrium with urea as a denaturant. The unfolding kinetics of these proteins were monitored during folding and unfolding by circular dichroism (CD) and fluorescence. During unfolding, CRABP II showed no intermediates, CRABP I had an intermediate with nativelike secondary structure, and CRBP II had an intermediate that lacked secondary structure. The refolding kinetics of these proteins were more similar. Each protein showed a burst-phase change in intensity by both CD and fluorescence, followed by a single observed phase by both CD and fluorescence and one or two additional refolding phases by fluorescence. The fluorescence spectral properties of the intermediate states were similar and suggested a gradual increase in the amount of native tertiary structure present for each step in a sequential path. However, the rates of folding differed by as much as 3 orders of magnitude and were slower than those expected from the contact order and topology of these proteins. As such, proteins with the same final structure may not follow the same route to the native state.