Saturation analysis of cellular retinoid binding proteins: application to retinoic acid resistant human neuroblastoma cells and to human tumors

A method for saturation analysis of cellular retinoic acid and retinol binding proteins, CRABP and CRBP, respectively, in cultured cells and human tumor samples, and its application to a retinoic acid resistant subline of the human neuroblastoma LA-N-5 cell line is described. Assessment of retinoid binding was accomplished by incubation of cytosols with increasing concentrations of [3H]retinoid (28-43 Ci/mmol; 1 Ci = 37 GBq) for 24 h. Bound retinoid was separated from free retinoid by adsorption with dextran-coated charcoal. Nonspecific binding was quantitated in parallel incubations which had been treated with p-chloromercuribenzene sulfonate (PCMBS), resulting in selective elimination of sulfhydryl-dependent ligand binding to both CRABP and CRBP. Quantitation was accomplished by Scatchard analysis of specific (PCMBS sensitive) binding. Employing this technique, specific retinoid binding was attributed to the presence of 2S macromolecules which displayed the known properties of CRABP and CRBP, namely ligand specificity, saturability, high ligand affinity, and PCMBS sensitivity. The apparent dissociation constants (Kd) for retinoic acid binding in cytosols prepared from murine 3T6 fibroblasts, rat testes, and a human ovarian tumor were 7, 11, and 35 nM, respectively. These preparations also bound retinol with high affinity, exhibiting Kds of 12, 26, and 48 nM, respectively. A retinoic acid resistant subline of LA-N-5 cells designated LA-N-5-R9 was established by long-term culture in the presence of 10(-6) M retinoic acid. This subline is resistant to the effects of retinoic acid in that it requires a 10-fold higher concentration of retinoic acid for 50% inhibition of growth than the parent line and displays no retinoic acid induced morphologic differentiation. Saturation analysis of CRABP in the parent and resistant subline reveal no significant alteration in either CRABP content or affinity. These results indicate that resistance to retinoic acid induced differentiation in LA-N-5-R9 occurs distal to CRABP binding or that CRABP does not mediate this response to retinoic acid.     

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.     

A high-performance liquid chromatographic technique that separates cellular retinol binding protein from cellular retinoic acid binding protein

A one-step procedure to detect cellular [3H]retinol and [3H]retinoic acid binding proteins (CRBP and CRABP) from rat testis cytosolic extract was devised. The procedure is based on anion-exchange high-performance liquid chromatography of the cytosolic fraction on columns of Mono Q, which permits elution of CRABP and CRBP at 12 and 22 min, respectively.     

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.     

Effects of dietary retinoic acid on cellular retinol- and retinoic acid-binding protein levels in various rat tissues

A study was conducted to explore the effects of retinoic acid, fed to retinol-deficient rats, on the tissue distribution and levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP). Sensitive and specific radioimmunoassays were employed to measure the levels of both CRBP and CRABP. Two groups of six male rats each were fed a purified retinoid-deficient diet supplemented with either: i) retinyl acetate (control group); or ii) retinoic acid (30 mg/kg diet) (retinol deficient-retinoic acid group). The retinoic acid supplementation was begun after 38 days on the retinoid-deficient diet alone, and was continued for 52-54 days. Analysis of the data indicated that only the CRBP level of the proximal epididymis in the retinol-deficient/retinoic acid group differed significantly from (was lower than) the corresponding control level, at the 1% confidence level. CRABP tissue levels did not differ significantly between the two groups. Thus, a moderately large intake of retinoic acid, as the only source of retinoids, had very little effect on the tissue distribution or levels of either its own cellular binding protein (CRABP) or of CRBP. This study provides further information showing that the tissue levels of the cellular retinoid-binding proteins are highly regulated and maintained in rats, even in the presence of marked changes in retinoid nutritional status.     

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.     

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.     

The effect of dibutyryl cyclic AMP and butyrate on F9 teratocarcinoma cellular retinoic acid-binding protein activity

F9 teratocarcinoma cells contain a cellular retinoic acid-binding protein (CRABP) that may mediate the retinoic acid-induced differentiation of this cell line. Specific [3H]retinoic acid binding to CRABP in F9 stem cell cytosol is protein-dependent, reaches equilibrium within 4 h at 4 degrees C, and yields 643 +/- 105 fmol of [3H]retinoic acid per mg of protein with an apparent dissociation constant of 9.2 +/- 1.1 nM. When F9 stem cells are grown in the presence of either dibutyryl cyclic AMP or sodium butyrate, CRABP activity is stimulated 2-4-fold. The effect of these drugs on CRABP activity is both time and concentration-dependent, resulting in an increase in the number of binding sites for [3H]retinoic acid with no change in their affinity. The new [3H]retinoic acid-binding sites have a sedimentation coefficient of 2 S and are not displaced by excess retinol. When F9 stem cells are grown in the presence of cyclic 8-bromo-AMP or cholera toxin, no increase in CRABP activity is observed. We conclude that the stimulation of CRABP activity by dibutyryl cyclic AMP may result from the action of butyrate. In addition, the stimulation of retinoic acid-induced F9 cell differentiation by cyclic AMP analogs (Strickland, S., Smith, K.K., and Marotti, K.R. (1980) Cell 21, 347-355) and the inhibition of this differentiation by butyrate (Levine R. A., Campisi, J., Wang, S.-Y., and Gudas, L. J. (1984) Dev. Biol. 105, 443-450) are not correlated with increases or decreases, respectively, in the level of CRABP activity.     

Lipid binding activities of the P2 protein in peripheral nerve myelin

Lipid binding activities of the P2 protein in peripheral nerve myelin were examined using retinoic acid, retinol and oleic acid as ligands. The P2 protein showed the specific binding affinity to both of retinoic acid and retinol. The binding site of these ligands was suggested to be similar. In addition, the high binding activity of the P2 protein with oleic acid was also observed. The ligands specificities of the P2 protein are clearly different from those of cellular retinoic acid binding protein (CRABP), cellular retinol binding protein (CRBP), and Z protein. In amino terminal sequence, however, the P2 protein contained considerable homologous structure to these lipid binding proteins. Therefore, the P2 protein and these lipid binding proteins may belong to a family of structurally related proteins evolved from a common ancestral gene.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

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.     

Analyses of the interactions between retinoid-binding proteins and embryonal carcinoma cells

[3H]Retinoic acid (RA) and [3H]retinol bind in an unsaturable manner to isolated nuclei from Nulli-SCC1 and PCC4.aza1R embryonal carcinoma (EC) cells. When nuclei are challenged with the same labeled retinoids on their respective binding proteins (CRABP and CRBP), much less binding is observed and the binding is saturable. RA-CRABP does not compete with [3H]retinol-CRBP for binding to specific Nulli-SCC1 nuclear sites, whereas retinol-CRBP (but not apo-CRBP) actually potentiates the binding of [3H]RA-CRABP to these nuclei. The binding of [3H]RA-CRABP and [3H]retinol-CRBP is not dramatically affected by prior removal of the outer nuclear membrane with Triton X-100. However, treatment with the detergent after the binding reaction is complete removes about half of the bound [3H]RA-CRABP and almost all of the bound [3H]retinol-CRBP. We measured specific retinoid-binding activities in nucleoplasmic extracts of Nulli-SCC1 and PCC4.aza1R cells. The only readily detectable specific binding activity in nucleoplasmic extracts from untreated cells was for [3H]retinol in PCC4.aza1R preparations. Nucleoplasmic extracts from Nulli-SCC1 and PCC4.aza1R cells pretreated with RA had considerable levels of specific [3H]RA-binding activity with little or no increase in [3H]retinol binding. By contrast, similar extracts from Nulli-SCC1 cells treated with retinol bound large amounts of both [3H]retinol and [3H]RA. Under the same conditions, PCC4.aza1R extracts also contained [3H]RA-binding activity with no increase in [3H]retinol binding above the high endogenous levels. Although these results might reflect translocation of binding proteins from cytoplasm to nucleus, other interpretations must be considered since we often observed an increase, rather than the expected reduction, in cytoplasmic retinoid-binding protein levels.     

Retinoid-binding proteins and retinol esterification in cultured retinal pigment epithelium cells

The esterification of all-trans retinol and the occurrence of cytosolic retinoid-binding proteins was investigated in cultured bovine retinal pigment epithelium (RPE) cells. ³H-labeled all-trans retinyl ester (mainly palmitate) was formed at an initial rate of 0.1 nmol·mg protein⁻¹·min⁻¹ when ³H-labeled all-trans retinol was incubated with the 100,000 g pellet obtained from a homogenate of freshly-harvested cells. No esterification could be detected under the same conditions after 14 days in culture in defined medium (DM) or in medium containing 20% fetal bovine serum (CM). No enhancement or restoration of esterifying capacity was observed when the assay mixture was supplemented with palmitoyl CoA. As determined by specific, saturable binding of ³H-labeled all-trans retinol and ³H-labeled 11-cis retinal to proteins with mol. wts 16,000 and 33,000 dalton on calibrated Bio-Sil TSK 250 size-exclusion columns, the cytosol of freshly-harvested RPE cells contained cellular retinol-binding protein (CRBP) and cellular retinal-binding protein (CRAlBP). By comparison with the quantity of ³H-labeled all-trans retinol bound under identical conditions to pure dog liver CRBP, it was estimated that fresh RPE cells contained 102 ± 3 ng CRBP·μg cytosol protein⁻¹. In cultured and subcultured cells, CRBP was present at much lower levels (down to one-tenth of the initial amounts) and CRAlBP could not be detected. Since binding of ³H-labeled all-trans retinoic acid to a protein with molecular weight of 17,000 dalton was not observed in the cytosols of fresh or cultured cells, it was concluded that cellular retinoic acid binding protein (CRABP) was either present at very low levels or absent altogether. An unidentified peak of specific ³H-labeled all-trans-retinoic acid binding at mol. wt 61,000 dalton was prominent in subcultured cells. These results show that in RPE cells in culture the expression of differentiated phenotype with respect to retinoid utilization undergoes significant modification. It is postulated that changes in the composition of the extracellular matrix (e.g. absence of interstitial retinol-binding protein, IRBP) may be involved.     

Influence of retinoid nutritional status on cellular retinol- and cellular retinoic acid-binding protein concentrations in various rat tissues

Studies were conducted to explore the effects of differences in retinoid nutritional status and of sex on the tissue distribution and levels of cellular retinol-binding protein (CRBP) and of cellular retinoic acid-binding protein (CRABP) in the rat. Sensitive and specific radioimmunoassays were developed and employed to measure the levels of both CRBP and CRABP. Four groups of six male rats each were fed experimental diets that differed greatly in the amount and kind of retinoids provided, but were otherwise identical. These groups were comprised of rats that were normal controls, retinoid-deficient, retinoic acid-fed, and excess retinol-fed. A fifth group of six female rats was fed the control diet. Immunogens identical with rat testis CRBP and CRABP, as assessed by radioimmunoassay displacement curves, were found in every rat tissue examined (21 tissues in males, 18 in females). The highest levels of CRBP were found in the proximal portion of the epididymis, the liver, and kidney. The highest levels of CRABP were found in the seminal vesicles, vas deferens, and skin. A significant (p less than 0.01) inverse relationship was found between CRBP and CRABP levels in the different tissues of the male reproductive tract. In both males and females, CRBP levels were highest in the gonads and proximal portion of the reproductive tract and decreased distally, whereas the opposite was true for CRABP. Retinoid-deficient rats showed reduced tissue levels of CRBP; thus, tissue CRBP levels are influenced by diet and retinoid availability. No differences in tissue CRBP levels were found in the rats fed the control, the retinoic acid, or the excess retinol diets. Female control rats had higher CRBP levels than male controls in 4 of 15 tissues compared (liver, lung, thymus, and fat). In contrast, tissue CRABP levels showed no diet- or sex-dependent differences. Only in one tissue, the skin, were differences observed (lower CRABP in retinoid-deficient and in female rats). Thus, CRABP metabolism and levels appear to be minimally influenced by the amount or kind of retinoid ligand available or by sex.     

Retinoids, retinoid-binding proteins, and retinyl palmitate hydrolase distributions in different types of rat liver cells

A study was conducted to determine the levels and distributions of retinoids, retinol-binding protein (RBP), retinyl palmitate hydrolase (RPH), cellular retinol-binding protein (CRBP), and cellular retinoic acid-binding protein (CRABP) in different types of isolated liver cells. Highly purified fractions of parenchymal, fat-storing (stellate), endothelial, and Kupffer cells were isolated in high yield from rat livers. The retinoid content of each fraction was measured by HPLC analysis. RBP, CRBP, and CRABP were measured by sensitive and specific radioimmunoassays, and RPH activity was measured by a sensitive microassay. The concentrations of each parameter expressed per 10(6) parenchymal or fat-storing cells were, respectively: retinoids, 1.5 and 83.9 micrograms of retinol equivalents; RBP, 138 and 7.4 ng; RPH, 826 and 1152 pmol FFA formed hr-1; CRBP, 470 and 236 ng; and CRABP, 5.6 and 8.7 ng. When these data were expressed on the basis of per unit mass of cellular protein, the concentrations of RPH, CRBP, and CRABP in the fat-storing cells, which contain 10-fold less protein than the large parenchymal cells, were seen to be greatly enriched over parenchymal cells. The parenchymal cells contained approximately 9% of the total retinoids, 98% of the total RBP, 90% of the total RPH activity, 91% of the total CRBP, and 71% of the total CRABP found in the liver. The fat-storing cells accounted for approximately 88% of the total retinoids, 0.7% of the total RBP, 10% of the RPH activity, 8% of the total CRBP, and 21% of the CRABP in the liver. The endothelial and Kupffer cell fractions contained very low levels of all of these parameters. Thus, the large and abundant parenchymal cells account for greater than 70% of the liver's RBP, RPH, CRBP, and CRABP; but the much smaller and less abundant fat-storing cells contain the majority of hepatic retinoids and greatly enriched concentrations of RPH, CRBP, and CRABP.     

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.     

Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein

We have investigated the esterification by liver membranes of retinol bound to cellular retinol-binding protein (CRBP). When CRBP carrying [3H]retinol as its ligand was purified from rat liver cytosol and incubated with rat liver microsomes, a significant fraction of the [3H]retinol was converted to [3H]retinyl ester. Esterification of the CRBP-bound [3H]retinol, which was maximal at pH 6-7, did not require the addition of an exogenous fatty acyl group. Indeed, when additional palmitoyl-CoA or coenzyme A was provided, the rate of esterification increased either very slightly or not at all. The esterification reaction had a Km for [3H]retinol-CRBP of 4 +/- 0.6 microM and a maximum velocity of 145 +/- 52 pmol/min/mg of microsomal protein (n = 4). The major products were retinyl palmitate/oleate and retinyl stearate in a ratio of approximately 2 to 1 over a range of [3H]retinol-CRBP concentrations from 1 to 8 microM. The addition of progesterone, a known inhibitor of the acyl-CoA:retinol acyltransferase reaction, consistently increased the rate of retinyl ester formation when [3H]retinol was delivered bound to CRBP. These experiments indicate that retinol presented to liver microsomal membranes by CRBP can be converted to retinyl ester and that this process, in contrast to the esterification of dispersed retinol, is independent of the addition of an activated fatty acid and produces a pattern of retinyl ester species similar to that observed in intact liver. A possible role of phospholipids as endogenous acyl donors in the esterification of retinol bound to CRBP is supported by our observations that depletion of microsomal phospholipid with phospholipase A2 prior to addition of retinol-CRBP decreased the retinol-esterifying activity almost 50%. Conversely, incubating microsomes with a lipid-generating system containing choline, CDP-choline, glycerol 3-phosphate, and an acyl-CoA-generating system prior to addition of retinol-CRBP increased retinol esterification significantly as compared to buffer-treated controls.     

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.     

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.