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.     

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.     

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.     

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.     

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)     

Polyomavirus small t antigen prevents retinoic acid-induced retinoblastoma protein hypophosphorylation and redirects retinoic acid-induced G0 arrest and differentiation to apoptosis

Polyomavirus small t antigen (ST) impedes late features of retinoic acid (RA)-induced HL-60 myeloid differentiation as well as growth arrest, causing apoptosis instead. HL-60 cells were stably transfected with ST. ST slowed the cell cycle, retarding G2/M in particular. Treated with RA, the ST transfectants continued to proliferate and underwent apoptosis. ST also impeded the normally RA-induced hypophosphorylation of the retinoblastoma tumor suppressor protein consistent with failure of the cells to arrest growth. The RA-treated transfectants expressed CD11b, an early cell surface differentiation marker, but inducible oxidative metabolism, a later and more mature functional differentiation marker, was largely inhibited. Instead, the cells underwent apoptosis. ST affected significant known components of RA signaling that result in G0 growth arrest and differentiation in wild-type HL-60. ST increased the basal amount of activated ERK2, which normally increases when wild-type cells are treated with RA. ST caused increased RARalpha expression, which is normally down regulated in RA-treated wild-type cells. The effects of ST on RA-induced myeloid differentiation did not extend to monocytic differentiation and G0 arrest induced by 1,25-dihydroxy vitamin D3, whose receptor is also a member of the steroid-thyroid hormone superfamily. In this case, ST abolished the usually induced G0 arrest and retarded, but did not block, differentiation without inducing apoptosis, thus uncoupling growth arrest and differentiation. In sum, the data show that ST disrupted the normal RA-induced program of G0 arrest and differentiation, causing the cells to abort differentiation and undergo apoptosis.     

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.     

Purification and partial characterization of cellular retinoic acid-binding protein from human placenta

Cellular retinoic acid-binding protein (CRABP) has been purified to homogeneity from human placenta by a series of procedures, including acetone powder extraction, gel filtration on Sephadex G-50, and ion-exchange chromatography on DEAE-cellulose and on SP-Sephadex. Cellular retinol-binding protein (CRBP) was isolated concurrently. CRABP was purified 75,400-fold, based on total soluble acetone powder extract of placenta. The protein is a single polypeptide chain with a molecular mass of 14,600 Da, estimated by sodium dodecyl sulfate (SDS) gel electrophoresis or gel filtration, and has an isoelectric point of 4.78 (apo-CRABP, 4.82). On analysis of absorption and fluorescence spectra, the protein was seen to exhibit an absorption peak at 350 nm, fluorescence excitation maxima at 350 and 370 nm, and a fluorescence emission maximum at 475 nm. Human CRABP was immunologically distinct from human CRBP and serum retinol-binding protein.     

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.     

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)