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.     

Distribution and levels of cellular retinol- and cellular retinoic acid-binding protein in various types of rat testis cells

The distribution and levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) were measured in rat testicular peritubular and Sertoli cells and in isolated rat pachytene spermatocytes and spermatids. Two Sertoli cell preparations, one containing some germ cells and another that had been osmotically shocked to destroy germ cells, were examined. CRBP and CRABP levels were measured by specific and sensitive radioimmunoassays. Testicular peritubular cell cytosol preparations were found to contain high levels of CRBP (1.48 +/- 0.87 microgram CRBP/mg protein) but CRABP could not be detected. The mean CRBP level in Sertoli cell preparations that contained some germ cells was 0.93 +/- 0.24 microgram CRBP/mg protein; this value was similar to the level of 1.11 +/- 0.20 microgram CRBP/mg protein measured for Sertoli cells free of germ cells. The level of CRABP found in Sertoli cell preparations containing germ cells (0.81 +/- 0.32 microgram CRABP/mg protein) was approximately five times greater than was observed in Sertoli cells free of germ cells (0.16 +/- 0.03 microgram CRABP/mg protein). CRBP and CRABP levels in cultured Sertoli cells were not affected by time in culture for up to five days of culture. Pachytene spermatocytes and spermatids were very enriched in CRABP (0.72 +/- 0.26 microgram CRABP/mg protein for spermatocytes and 0.65 +/- 0.21 microgram CRABP/ml protein for spermatids). A search for a high molecular weight retinol-binding protein did not demonstrate the existence of such a protein in Sertoli cell-conditioned medium. In summary, these studies provide quantitative information about the distribution of the cellular retinoid-binding proteins in the cell types that compose the rat testis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular retinoic acid-binding protein messenger RNA: levels in rat tissues and localization in rat testis.

Studies were conducted to explore the tissue- and cell-specific regulation of cellular retinoic acid-binding protein (CRABP) expression in the rat. Two studies were carried out. The first explored the regulation of CRABP mRNA levels in selected rat tissues by dietary retinoid status, and the relationship between CRABP mRNA and protein levels in different tissues. The second examined the cellular localization of CRABP expression in the testis. In order to conduct these experiments, a cDNA encoding CRABP was isolated and characterized. The DNA sequence of the coding region had 96% identity with that of the mouse CRABP cDNA and encodes a protein identical to mouse and bovine CRABP. CRABP mRNA and protein levels were quantified in five tissues from normal, retinoid-deficient, and retinol-repleted rats. Tissue CRABP and CRABP mRNA levels were highly correlated (P less than 0.01) indicating that inter-tissue variability of CRABP levels mainly results from regulation of CRABP mRNA levels. Neither CRABP protein nor mRNA levels were affected by retinol deficiency, in marked contrast with results previously demonstrated with cellular retinol-binding protein (CRBP) (J. Lipid Res. 1990. 31: 821-829). 35S-labeled CRABP cRNA probes were used to localize CRABP mRNA within the testis of adult rats by in situ hybridization. CRABP mRNA was localized selectively in the periphery of the seminiferous tubules, primarily in type A spermatogonia. The localization of CRABP mRNA differs from that of CRABP protein, which is known to be enriched in maturing and more mature germinal cells. This difference suggests that CRABP in germ cells may be highly stable, remaining in the maturing germ cells without degradation long after CRABP mRNA levels have declined to very low levels. The specific localization of CRABP mRNA and protein presumably reflects the biological roles of retinoic acid in the development and/or later function of germinal cells.     

Purification and properties of cellular retinoic acid-binding protein from neonatal rat skin

Cellular retinoic acid-binding protein (CRABP) was detected in cytosolic extracts of dermis and epidermis of neonatal rat skin using high-performance size-exclusion liquid chromatography and was more abundant in dermal tissue. CRABP was purified 1000-fold from an acid-precipitated, 50,000 x g supernatant of neonatal rat skin by ion-exchange chromatography on DEAE-Sephacel, followed by chromatofocussing and hydrophobic-interaction chromatography. The protein had an apparent Mr of 14,800. In chromatofocussing experiments the apoprotein and holoprotein gave different elution profiles, indicating a charge difference between the two forms. The ability of various retinoids to compete with all-trans-retinoic acid for binding to CRABP was assayed: 4-oxoretinoic acid and two synthetic retinoids were effective competitors, but 13-cis-retinoic acid, 3,4-didehydroretinoic acid and the acid derivative of etretinate competed poorly. The binding protein had a Kd for all-trans-retinoic acid of 8 nM using a dextran-charcoal assay, but a higher value was obtained using high-performance size-exclusion liquid chromatography. The holoprotein dissociated rapidly at room temperature and had a half-life of 4.7 min. At 0 degrees C, the holoprotein had a half-life of 200 min.     

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.     

Cellular localization of mRNAs for retinoic acid receptor-alpha, cellular retinol-binding protein, and cellular retinoic acid-binding protein in rat testis: evidence for germ cell-specific mRNAs

In the present study we have examined the cellular localization and developmental changes of mRNAs for retinoid-binding proteins in rat testis. We demonstrate that mRNA (0.7 kb) for cellular retinol-binding protein (CRBP) is expressed only in Sertoli cells and peritubular cells. The mRNA for CRBP could not be detected in other testicular cells. In contrast, mRNA for cellular retinoic acid-binding protein (CRABP) was detected primarily in germ cells and to a small extent in tumor Leydig cells. The mRNA for CRABP in germ cells revealed distinct size heterogeneity and three distinct mRNA species were observed (1.0, 1.8, and 1.9 kb), in contrast to previous data for somatic cells where only the 1.0-kb mRNA has been reported. Messenger RNAs for retinoic acid receptor-alpha (RAR alpha) were detected in both somatic and haploid germ cells. The highest level of RAR alpha was seen in Sertoli cells, round spermatids, and tumor Leydig cells. Lower, but distinct, levels were observed in peritubular cells. Furthermore, we observed germ cell-specific species of RAR alpha mRNA (4 kb and approximately 7 kb). The smallest mRNA for RAR alpha (2.7 kb) in somatic cells was absent in germ cells. The levels of mRNAs for the various retinoid-binding proteins in whole testis obtained from rats of various ages confirmed this cellular localization. The mRNAs for CRBP, the small molecular size (2.7 kb) mRNA for RAR alpha (localized to somatic cells), and the 1-kb mRNA for CRABP showed an age-dependent decrease.(ABSTRACT TRUNCATED AT 250 WORDS)     

The primary structure of bovine cellular retinoic acid-binding protein

The complete amino acid sequence of bovine adrenal gland cellular retinoic acid-binding protein (CRABP) has been determined. The primary structure was established by analyses of cyanogen bromide fragments and peptides obtained by trypsin and Staphylococcus aureus protease digestions. The polypeptide chain of bovine CRABP comprises 136 amino acid residues. From partial sequence information, CRABP has been shown to be homologous to cellular retinol-binding protein, myelin protein P2, and the fatty acid-binding Z-protein. A comparison of the complete amino acid sequences of the members of this protein family, which also includes the rat intestinal fatty acid-binding protein, shows that CRABP is more similar to cellular retinol-binding protein and protein P2 than to the fatty acid-binding proteins. All five proteins are very similar in their NH2-terminal regions, suggesting that this part is important for a property common to the members of this protein family. This is the first report of a complete amino acid sequence of a CRABP.     

Purification and characterization of a murine cellular retinoic acid-binding protein

A cellular retinoic acid-binding protein from 1-day-old mouse pups has been purified to homogeneity. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE cellulose, and chromatofocusing on PBE9-4 ion exchange resin. The chromatofocusing step was most useful in removing the major contaminants, which were otherwise difficult to remove. The binding protein was finally subjected to two cycles of high performance liquid chromatography on a DEAE-5PW column to achieve homogeneity. The protein has an isoelectric point of 4.75 and consists of a single polypeptide, migrating with an apparent Mr of 14,600 in SDS--polyacrylamide gel electrophoresis. Amino-terminal sequence analysis showed that the mouse cellular retinoic acid-binding protein has a high percentage of amino acid identity with other retinoid-binding proteins. However, it is immunologically distinct from the cellular retinol-binding protein.     

Quantitation and tissue localization of the cellular retinoic acid-binding protein

The distribution of the cellular retinoic acid-binding protein (CRABP) in some rat tissues has been determined, and the protein has been localized by immunocytochemical techniques in sections from rat testis. In the testis CRABP was found in the seminiferous tubuli with Sertoli cells and the spermatogonia most intensely stained. All other cells of the germinal epithelium appeared largely devoid of CRABP. By use of an enzyme-linked immunosorbent assay CRABP was quantitatively estimated in several tissues and the highest levels were found in testis and eye. Comparisons of the tissue levels of CRABP and of the cellular retinol-binding protein (CRBP) did not reveal any apparent correlation.     

The complete amino acid sequence of the cellular retinoic acid-binding protein from bovine retina

The complete amino acid sequence of cellular retinoic acid-binding protein from bovine retina was obtained by Edman degradation of peptides obtained from enzymatic and CNBr digests. The 136 residue sequence is identical to that reported recently for the protein from bovine adrenal tissue indicating that the same gene is expressed in both tissues.     

Upregulation of cellular retinoic acid-binding protein I expression by ethanol

Acute and chronic ethanol ingestion cause embryopathy similar to that of hyper- or hypovitaminosis A. Experimental data have suggested interaction between vitamin A and alcohol signaling pathways at the level of metabolic interference, which ultimately affects the concentration of retinoic acid (RA) in animals. The present study was set up to examine the possible effects of alcohol on cellular RA binding protein I (CRABP-I) expression during embryonic development by using transgenic mouse embryos and P19 embryonal carcinoma cells as experimental models. It was found that expression of the mouse CRABP-I gene was elevated in developing embryos at mid-gestation stages as a result of ethanol consumption by the mothers. Specific elevation of this gene was detected in the limb bud and the gut. In the P19 model, the CRABP-I gene was directly upregulated by ethanol, which was not blocked by a protein synthesis inhibitor. Furthermore, the regulation of the CRABP-I gene by ethanol was mediated by the 5' upstream regulatory region of the CRABP-I gene promoter. A potential interaction of vitamin A and ethanol at the level of CRABP-I gene expression is discussed.     

Retinoids and cultured human fibroblasts : Effects on cell growth and presence of cellular retinoic acid-binding protein

We have examined the effects of retinoids on growth of cultured human skin fibroblasts from four individuals. Retinoic acid and retinol both produce a dose-dependent inhibition of growth in the four strains examined; retinoic acid was more potent than retinol in this respect. The growth inhibitory effect of retinoic acid is characterized by a decrease in the exponential growth rate, which is reversible upon removal of retinoic acid from the growth medium; the final saturation density, however, is not modified by retinoic acid treatment. No alterations of cell morphology, viability, or adhesiveness to substratum are induced by the retinoid concentrations utilized. The inhibitory effect of 10⁻⁶ M retinoic acid on cell growth is not affected by the concentration of fetal calf serum (FCS) in the medium. In all four human fibroblast strains examined, specific binding of [³H]retinoic acid to cytosol is present as determined by sucrose-density gradient centrifugation. Despite the effects of retinol on fibroblast growth, no cytoplasmic binding of [³H]retinol could be demonstrated in these cells.     

Purification and properties of retinol- and retinoic acid-binding proteins from a transplantable mouse colon tumor

Cellular retinol-binding protein and retinoic acid-binding protein, the possible mediators of the action of retinoids in epithelial differentiation and control of tumorigenesis, have been reproducibly purified from mouse colon tumor 26, and some of their properties were studied. The main steps of purification involved acid-precipitation, DEAE-Sephadex, CM-cellulose and Sephadex G-100 chromatography. About 2 mg of the binding proteins were isolated from 60 g tumor. The purified preparations showed only two protein bands on polyacrylamide gel electrophoresis. The two binding proteins were partially resolved by sedimentation equilibrium technique; but was completely separable by preparative electrophoresis in the presence of sodium dodecyl sulfate. The retinol- and retinoic acid-binding proteins are presumably monomers with molecular weights of 15,500 and 14,600, respectively, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. On gel filtration however, both the binding proteins retarded to the same molecular size of 17,800. On preparative columns, both the proteins expressed the same isoelectric pH, 4.5. Both proteins of the tumor possessed functional thiol groups. The mercurial inhibition of the binding capacity of the proteins for their ligands was reversible upon treatment with thiol compounds.