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.     

Use of retinoids to analyze the cellular basis of positional memory in regenerating amphibian limbs

Cells of the amphibian limb regeneration blastema inherit memories of their level of origin (positional memory) along the limb axes. These memories serve as boundaries of what is to be regenerated, thus preventing regeneration of any but the missing structures. Because of its importance in determining the boundaries of regenerate pattern, it is essential to understand the cellular and molecular basis of positional memory. One approach to this problem is to look for position-related differences in a cell or molecular property along a limb axis and then show, using an agent that modifies regenerate pattern, that the cell or molecular property and the pattern are coordinately modified. We have done this using retinoic acid (RA) as a pattern-modifying agent and an in vivo assay that detects position-related differences in a cell recognition-affinity property along the proximodistal (PD) axis of the regenerating axolotl limb. RA proximalizes positional memory in the PD axis, posteriorizes it in the anteroposterior axis, and ventralizes it in the dorsoventral axis. The level-specific PD cell recognition-affinity property is proximalized by RA, indicating that this property and positional memory are causally related. The effects of RA on positional memory may be mediated through a cellular RA-binding protein (CRABP), since the concentration of unbound (apo) CRABP molecules is highest during early stages of regeneration when the proximalizing effects of RA are greatest.     

Expression of cellular retinoic acid binding protein (CRABP) in Escherichia coli. Characterization and evidence that holo-CRABP is a substrate in retinoic acid metabolism.

Cellular retinoic acid binding protein (CRABP) has been expressed efficiently in Escherichia coli from the cDNA of bovine adrenal CRABP and characterized, especially with respect to affinity for endogenous retinoids and a role for it in retinoic acid metabolism. The purified E. coli-expressed CRABP was similar to authentic mammalian CRABP in molecular weight (approximately 14,700), isoelectric point (4.76), absorbance maxima (apo-CRABP, 280 nm; holo-CRABP, 350 and 280 nm with the ratio A350/A280 = 1.8), and in fluorescence excitation (350 nm) and emission spectra (475 nm). The equilibrium dissociation constant, Kd, of E. coli-derived CRABP and all-trans-retinoic acid was 10 +/- 1 nM (mean +/- S.D., n = 4) by retinoid fluorescence and 7 +/- 1 nM (mean +/- S.D., n = 3) by quenching of protein fluorescence, but neither retinol nor retinal bound in concentrations as high as 7 microM. All-trans-cyclohexyl ring derivatives of retinoic acid (3,4-didehydro-, 4-hydroxy-, 4-oxo-, 16-hydroxy-4-oxo-, 18-hydroxy-) had affinities similar to that of all-trans-retinoic acid, whereas 13-cis-retinoic acid and 4-oxo-13-cis-retinoic acid had approximately 25-fold lower affinity. Holo-CRABP was a substrate for retinoic acid catabolism in rat testes microsomes by three criteria: 1) the rate of retinoic acid metabolism with CRABP in excess of retinoic acid exceeded the rate supported by the free retinoic acid; 2) increasing the apo-CRABP did not decrease the rate as predicted if free retinoic acid were the only substrate; and 3) holo-CRABP had a lower Michaelis constant (1.8 nM) for retinoic acid elimination than did free retinoic acid (49 nM). These data indicate a direct role for CRABP in retinoic acid metabolism and suggest a mechanism for discriminating metabolically between all-trans- and 13-cis-retinoids.     

The relationship among retinoid structure, affinity for retinoic acid-binding protein, and ability to respecify pattern in the regenerating axolotl limb

To further our understanding of the action of retinoids on the respecification of pattern in the regenerating axolotl limb we have studied the relative potencies of a range of synthetic and natural retinoids administered locally to the blastema. Alterations in the polar end group of the retinoic acid (RA) molecule to produce esters, the alcohol, or the aldehyde abolish the ability of the molecule to respecify pattern. On the other hand, alterations of the ring or side chain to produce the synthetic retinoids arotinoid and TTNPB considerably increases the potency of the molecule to respecify pattern--TTNPB is at least 100X more potent than retinoic acid. To examine the role of cellular retinoic acid-binding protein (CRABP) in the respecification process we determined the relative binding affinities of these retinoids for CRABP. These data correlated well with the respecification series: retinoids which showed no affinity for CRABP did not respecify pattern and those which did show affinity for CRABP did respecify pattern. Furthermore the most potent retinoid, TTNPB, has a higher affinity for CRABP than RA itself. This suggests that CRABP may be playing an important role in the action of RA on pattern formation in the regenerating limb.     

Affinity purification of cellular retinoic acid-binding protein on 14-carboxy-13-cis-retinamide-sepharose 4B.

A biologically active bifunctional retinoid, ethyl 14-carboxyretinoate, has been synthesized and shown to bind cellular retinoic acid (RA)-binding protein (CRABP) via its free carboxy group. We describe herein the synthesis of 14-carboxy-13-cis-retinamide-Sepharose 4B, which is an affinity matrix bearing an all-trans-RA moiety, and thus was used to purify and characterize CRABP from chick-embryo skin. An amide bond was first formed between the free carboxy group of the retinoid and a primary amino group of aminohexyl-Sepharose 4B, by reaction with carbodi-imide, and the ester group of the resin-bound retinoid was then hydrolysed in an alkaline medium. Polyacrylamide-gel electrophoresis and f.p.l.c. Superose column-chromatographic analysis demonstrated that the affinity-purified CRABP (Mr 15,000) was close to electrophoretic homogeneity (greater than 90%) and specifically interacts with RA. By using affinity gel chromatography, conversion of holo-CRABP into apo-CRABP by treatment with p-hydroxymercuribenzoate and a possible involvement of a thiol group in RA binding to CRABP were established. This affinity procedure provides several advantages: (i) 14-carboxy-13-cis-retinamide-Sepharose exhibited high efficiency and selectivity for RA-binding protein (i.e. retinol- or fatty-acid-binding proteins did not bind); (ii) the presence of the amide linkage between the ligand and the matrix makes this affinity resin relatively stable to cytosolic enzymes; and (iii) other RA-binding proteins, e.g. nuclear receptor(s), may be purified.     

Retinoic acid-binding protein in the axolotl: distribution in mature tissues and time of appearance during limb regeneration

Analysis of cytoplasmic protein preparations from axolotl tissues revealed the presence of a cytoplasmic retinoic acid-binding protein (CRABP), of approximate molecular weight 17K. This protein was found to be present at various concentrations in skin, muscle, and limb tissue preparations, but not in liver and serum preparations. The distribution and molecular weight of this protein agrees with that reported in mammalian studies. The level of CRABP in cone stage blastemas was found to be significantly higher than that found in nonregenerating whole limb preparations. The level falls gradually, to approach normal, towards the completion of regeneration. Such an increase, at the start of regeneration, was not altered by 4 days pretreatment with 36 mg/liter all-trans-retinoic acid, a sufficient dose to produce pattern effects. Competition experiments confirmed that the all-trans and 13-cis isomers of retinoic acid bind to CRABP with similar high efficiencies, and that the arotinoid, Ro 13-6298, exhibits only a fraction of this binding activity. Retinol, retinol palmitate, and retinol acetate were unable to compete with [3H]retinoic acid for binding to CRABP. The results presented here are discussed in terms of their possible value to understanding pattern specification in the regenerating urodele limb.     

视黄酸对赤子爱胜蚓再生头尾轴形成的影响

为了探讨视黄酸对蚯蚓再生的影响,用视黄酸处理了从不同部位剪切的蚯蚓体段．观察其存活率、重量和再生长度的变化。结果表明,有头无尾的体段存活率受视黄酸影响较小,而无头有尾的处理受视黄酸影响较大;视黄酸处理后30d,各处理再生长度和存活率均小于对照;视黄酸对蚯蚓再生有明显影响,能延迟和干扰再生,影响头部的形成。视黄酸影响蚯蚓再生的作用方式可能是通过干扰前后体轴的形成,从而影响蚯蚓再生图式形成。     

Expression and functional analysis of the cellular retinoic acid binding protein from silkworm pupae (Bombyx mori)

Cellular retinoic acid binding protein (CRABP) is a member of intracellular lipid-binding protein (iLBP), and closely associated with retinoic acid (RA) activity. We have cloned the CRABP gene from silkworm pupae and studied the interaction between Bombyx mori CRABP (BmCRABP) and all-trans retinoic acid (atRA). The MTT assay data indicated that when BmCRABP is overexpressed in Bm5 cells, the cells dramatically resisted to atRA-induced growth inhibition. Conversely, the cells were sensitive to atRA treatment upon knocking down the BmCRABP expression. Subcellular localization revealed that BmCRABP is a cytoplasm protein, even when treated with atRA, the CRABP still remained in the cytoplasm. These data demonstrated that the function of BmCRABP have an effect on the physiological function of atRA.     

Expression of cellular retinoic acid-binding protein I and II (CRABP I and II) in embryonic mouse hearts treated with retinoic acid

Cellular retinoic acid binding proteins are considered to be involved in retinoic acid (RA) signaling pathways. Our aim was to compare the expression and localization of cellular retinoic acid binding proteins I and II (CRABP I and II) in embryonic mouse hearts during normal development and after a single teratogenic dose of RA. Techniques such as real-time PCR, RT-PCR, Western blots and immunostaining were employed to examine hearts from embryos at 9-17 dpc. RA treatment at 8.5dpc affects production of CRABP I and II in the heart in the 48-h period. Changes in expression of mRNA for retinaldehyde dehydrogenase II (Raldh2), Crabp1 and Crabp2 genes also occur within the same time window (i.e. 10-11dpc) after RA treatment. In the embryonic control heart these proteins are localized in groups of cells within the outflow tract (OT), and the atrioventricular endocardial cushions. A gradient of labeling is observed with CRABP II but not for CRABP I along the myocardium of the looped heart at 11 dpc; this gradient is abolished in hearts treated with RA, whereas an increase of RALDH2 staining has been observed at 10 dpc in RA-treated hearts. Some populations of endocardial endothelial cells were intensively stained with anti-CRABP II whereas CRABP I was negative in these structures. These results suggest that CRABP I and II are independently regulated during heart development, playing different roles in RA signaling, essential for early remodeling of the heart tube and alignment of the great arteries to their respective ventricles.     

Localization of specific retinoid-binding sites and expression of cellular retinoic-acid-binding protein (CRABP) in the early mouse embryo

Retinoids (vitamin A derivatives) are important for normal embryogenesis and retinoic acid, an acidic derivative of vitamin A, was recently proposed to be an endogenous morphogen. Several retinoids are also potent teratogens. Using an autoradiographic technique, we have identified tissues and cells in early mouse embryos that are able to specifically accumulate a radiolabelled synthetic derivative of retinoic acid. Strong accumulation of radioactivity was seen in several neural crest derivatives and in specific areas of the CNS. Gel filtration analyses of cytosols from embryos that received the radiolabelled retinoid in utero suggested that cellular retinoic acid-binding protein (CRABP) was involved in the accumulation mechanism. Immunohistochemical localization confirmed that cells accumulating retinoids also expressed CRABP. Strong CRABP immunoreactivity was found in neural crest-derived mesenchyme of the craniofacial area, in visceral arches, in dorsal root ganglia and in cells along the gut and the major vessels of the trunk region. In CNS, CRABP expression and retinoid binding was largely restricted to the hindbrain, to a single layer of cells in the roof of the midbrain and to cells in the mantle layer of the neural tube. Our data suggest that cells in the embryo expressing CRABP are target cells for exogenous retinoids as well as endogenous retinoic acid. Retinoic acid may thus play an essential role in normal development of the CNS and of tissues derived from the neural crest. We propose that the teratogenic effects of exogenous retinoids are due to an interference with mechanisms by which endogenous retinoic acid regulates differentiation and pattern formation in these tissues.     

Nuclear interactions of retinoic acid-binding protein in chemically induced mammary adenocarcinoma.

Cellular retinoic acid-binding protein (CRABP) was detected in the nuclear fraction of N-methyl-N-nitrosourea-induced mammary cancers after the incubation of cytosol containing [3H]retinoic acid (RA)-bound CRABP with isolated nuclei. CRABP extracted from the nuclei in buffer containing 0.4 M-KCl sedimented as a 2 S component when subjected to sucrose-density-gradient analysis. [3H]RA-CRABP was found to be a prerequisite for the detection of nuclear binding, since the incubation of isolated nuclei or 0.4 M-KCl extract of the nuclei with [3H]RA did not result in any significant binding. Incubation of [3H]RA-CRABP at 25 or 30 degrees C before incubation with the nuclei neither altered the sedimentation coefficient nor enhanced the nuclear binding compared with 0 degrees C incubation. The tumour nuclei contained a saturable number of binding sites with a dissociation constant of 1.6 x 10(-9) M. These results indicate that the action of retinoic acid in the target organ may be mediated by its interaction with the nuclei.     

Cellular retinoic acid-binding protein II gene expression is directly induced by estrogen,but not retinoic acid,in rat uterus

It has been suggested that cellular retinoic acid-binding protein (II) (CRABP(II)) may have a role in the movement of retinoic acid (RA) to its nuclear receptors, thereby enhancing the action of RA in the cells in which it is expressed. RA has also been shown to increase expression of CRABP(II). Previous work from our laboratory has shown that 17 beta-estradiol (E2) administration to prepubertal female rats leads to acquisition of the ability of the lining epithelium to synthesize RA as well as to express CRABP(II). To determine whether this appearance of CRABP(II) was dependent on the production of RA, both E2 and RA were administered to ovariectomized rats. E2 administration induced expression of the CRABP(II) gene in the uterus within 4 h, and this induction was not inhibited by prior administration of puromycin, indicating that the induction was direct. In contrast, RA caused no change in CRABP(II) message level, even at times as late as 48 h after administration. Isolation and analysis of 4.5 kb of the 5'-flanking region of the gene revealed no apparent E2-response element. Using this portion of the gene to drive expression of the luciferase gene in transfected cells allowed identification of a region containing an imperfect estrogen-response element and estrogen-response element half-site, necessary for E2-driven induction. A possible Sp1 binding site in the 5'-flanking region of the CRABP(II) gene was also required for this induction. The ability of E2 to induce expression of CRABP(II) suggests that it can enhance the activity of RA, directly affecting expression of retinoid-responsive genes.