Retinol uptake and metabolism,and cellular retinol binding protein expression in an in vitro model of hepatic stellate cells

Liver is a major site of retinoid metabolism and storage, and more than 80% of the liver retinoids are stored in hepatic stellate cells. These cells represent less than 1% of the total liver protein, reaching a very high relative intracellular retinoid concentration. The plasma level of retinol is maintained close to 2 M, and hepatic stellate cells have to be able both to uptake or to release retinol depending upon the extracellular retinol status. In view of their paucity in the liver tissue, stellate cells have been studied in primary cultures, in which they loose rapidly the stored lipids and retinol, and convert spontaneously into the activated myofibroblast phenotype, turning a long-term study of their retinol metabolism impossible. We have analyzed the retinol metabolism in the established GRX cell line, representative of stellate cells. We showed that this cell line behaves very similarly, with respect the retinol uptake and release, to primary cultures of hepatic stellate cells. Moreover, we showed that the cellular retinol binding protein (CRBP-I) expression in these cells, relevant for both uptake and esterification of retinol, responds to the extracellular retinol status, and is correlated to the retinol binding capacity of the cytosol. Its expression is not associated with the overall induction of the lipocyte phenotype by other agents. We conclude that the GRX cell line represents an in vitro model of hepatic stellate cells, and responds very efficiently to wide variations of the extracellular retinol status by autonomous controls of its uptake, storage or release.     

Regulation of mRNA levels for cellular retinol binding protein in rat Sertoli cells by cyclic AMP and retinol

The levels of mRNA for cellular retinol binding protein (CRBP) were studied in primary rat Sertoli cell cultures treated with cAMP analogues and retinol. In the presence of cyclic AMP analogues a dose- and time-dependent reduction (70-90%) of the levels of mRNA for CRBP was observed. Retinol concentrations above 10 nM induced a dose- and time-dependent increase (2-3 fold) in mRNA levels for CRBP. Assuming that CRBP is important for vitamin A action, our data indicate that both cAMP and retinol itself modulate the sensitivity of the Sertoli cells for retinol.     

Partial characterization of nuclear binding sites for retinol delivered by cellular retinol binding protein

Retinol (vitamin A alcohol), which plays an important role in the differentiation of epithelia, can be transferred to chromatin in vitro. Rat liver chromatin can accept retinol in a specific and saturable manner only when the retinol is presented as a complex with cellular retinol-binding protein (CRBP). A partial characterization of the nuclear components responsible for accepting retinol is reported here. A preparation of solubilized chromatin isolated from liver nuclei was able to accept retinol from its complex with CRBP as described previously for nuclei and chromatin. The binding of retinol to chromatin was noncovalent. However, chromatin prepared from nuclei which were incubated with DNase I or micrococcal nuclease did not accept retinol specifically. Chromatin in the form of mono and dinucleosomes also did not accept retinol. However, treatment of nuclei with RNase did not affect the specific binding of retinol. Furthermore, it has been found that retinol was not transferred to purified double or single stranded DNA. These results are interpreted to indicate that the transfer of retinol to specific nuclear binding sites requires a higher order of chromatin structure than that occurring in nucleosome preparations.     

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.     

Retinol binding protein-albumin domain III fusion protein deactivates hepatic stellate cells

Liver fibrosis is characterized by accumulation of extracellular matrix, and activated hepatic stellate cells (HSCs) are the primary source of the fibrotic neomatrix and considered as therapeutic target cells. We previously showed that albumin in pancreatic stellate cells (PSCs), the key cell type for pancreatic fibrogenesis, is directly involved in the formation of vitamin A-containing lipid droplets, inhibiting PSC activation. In this study, we evaluated the anti-fibrotic activity of both albumin and retinol binding protein-albumin domain III fusion protein (R-III), designed for stellate cell-targeted delivery of albumin III, in rat primary HSCs and investigated the underlying mechanism. Forced expression of albumin or R-III in HSCs after passage 2 (activated HSCs) induced lipid droplet formation and deactivated HSCs, whereas point mutations in high-affinity fatty acid binding sites of albumin domain III abolished their activities. Exogenous R-III, but not albumin, was successfully internalized into and deactivated HSC-P2. When HSCs at day 3 after plating (pre-activated HSCs) were cultured in the presence of purified R-III, spontaneous activation of HSCs was inhibited even after passage 2, suggestive of a potential for preventive effect. Furthermore, treatment of HSCs-P2 with R-III led to a significant reduction in both cytoplasmic levels of all-trans retinoic acid and the subsequent retinoic acid signaling. Therefore, our data suggest that albumin deactivates HSCs with reduced retinoic acid levels and that R-III may have therapeutic and preventive potentials on liver fibrosis.     

Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor

STRA6 is a multitransmembrane domain protein not homologous to any other proteins with known function. It functions as the high-affinity receptor for plasma retinol binding protein (RBP) and mediates cellular uptake of vitamin A from the vitamin A-RBP complex. Consistent with the diverse roles of vitamin A and the wide tissue expression pattern of STRA6, mutations in STRA6 are associated with severe pathological phenotypes in humans. The structural basis for STRA6's biochemical function is unknown. Although computer programs predict 11 transmembrane domains for STRA6, its topology has never been studied experimentally. Elucidating the transmembrane topology of STRA6 is critical for understanding its structure and function. By inserting an epitope tag into all possible extracellular and intracellular domains of STRA6, we systematically analyzed the accessibility of each tag on the surface of live cells, the accessibility of each tag in permeabilized cells, and the effect of each tag on RBP binding and STRA6-mediated vitamin A uptake from the vitamin A-RBP complex. In addition, we used a new lysine accessibility technique combining cell-surface biotinylation and tandem-affinity purification to study a region of the protein not revealed by the epitope tagging method. These studies not only revealed STRA6's extracellular, transmembrane, and intracellular domains but also implicated extracellular regions of STRA6 in RBP binding.     

Isolation and purification of cellular retinol binding protein (CRBP) from goat mammary gland

The cellular retinol binding protein (CRBP) from goat mammary gland has been isolated in pure form. The molecular weight of CRBP by gel filtration was 15,120 dalton. The 280/350 absorbance ratio of this protein was 1.20 and it gave a single precipitin line on immunodiffusion. The method is very simple and reproducible.     

Regulation of cellular retinol binding protein type II by 1,25-dihydroxyvitamin D3.

Previously we purified and sequenced an 18-kDa chick duodenal protein that was modulated by 1,25-dihydroxyvitamin D3. The N-terminus of this protein has striking sequence homology to cellular retinol binding protein type II (CRBP II). Furthermore, this purified chick protein binds retinol. Antibodies have now been generated to the chick protein and used for immunoblot analysis to demonstrate that the chick protein has molecular weight, tissue distribution, and subcellular localization similar to rat CRBP II. These antibodies also cross-reacted with rat CRBP II. Antibodies to rat CRBP II cross-react with the chick protein. Northern analysis using a cDNA probe for rat CRBP II showed a single 860 base pair mRNA in both chick and rat intestinal RNA preparations. These results demonstrate that the 1,25-dihydroxyvitamin D3 modulated protein in chick embryonic organ culture is chick CRBP II. Pulse-chase experiments in chick embryonic duodenal organ culture strongly suggest that 1,25-dihydroxyvitamin D3 markedly decreases the synthesis of CRBP II, while not changing the degradation rate. The concentration of 1,25-dihydroxyvitamin D3 required for the decrease in CRBP II synthesis is approximately that required to stimulate calcium uptake into embryonic chick duodenal organ cultures.     

Retinol binding protein 4 levels relate to the presence and severity of coronary artery disease

BackgroundThe previous studies have showed that serum retinol binding protein 4 (RBP4) levels increase in metabolic disorders which are closely associated with cardiovascular diseases (CVD). However, the human studies investigating the role of RBP4 in CVD are conflicted. Therefore, we aimed to evaluate the relationship between RBP4 with the presence and severity of coronary artery disease (CAD) in this study.Methods55 patients with presenting acute coronary syndrome (ACS) and 43 control subjects who had various cardiovascular risk factors with normal coronary artery on coronary angiography were included in this study. The serum RBP4 concentrations were measured using ELISA method, clinically and anatomically score models were used to assess the severity of coronary lesion.ResultsSerum RBP4 levels were significantly higher in patients with ACS compared to the without ACS (68.40 ± 47.94 mg/L vs. 49.46 ± 13.64 mg/L; p = 0.014). RBP4 was correlated with GENSINI and SYNTAX I score (r = 0.286 p = 0.034; r = 0.403 p = 0.002 respectively). However, there was no relationship between RBP4 and GRACE score.ConclusionsThe serum RBP4 levels increase in patients with CAD and its increased levels may be correlated with CAD severity.     

The cellular retinol binding protein II gene. Sequence analysis of the rat gene, chromosomal localization in mice and humans, and documentation of its close linkage to the cellular retinol binding protein gene

Cellular retinol binding protein II (CRBP II) is an abundant, 134-residue protein present in the small intestinal epithelium. It is thought to participate in the uptake and/or intracellular metabolism of vitamin A. We have isolated and sequenced the rat CRBP II gene. Its four exons span 0.65 kilobases and are interrupted by three introns with an aggregate length of 19.5 kilobases. Southern blot hybridization analysis indicated that this gene is highly conserved in rats, mice, and humans. CRBP II belongs to a protein family that contains eight known members. Computer-assisted comparative sequence analyses indicated that a region of internal homology spans its first two exons and that oligopeptide domains specified by these first two exons exhibit significant homology to all other family members as well as to a portion of the all-trans-retinol binding domain that has previously been defined in serum retinol binding protein. The CRBP II gene was mapped in mice using recombinant inbred strains and restriction fragment length polymorphisms. It is located on chromosome 9 within 5.3 centimorgans of the phosphoglucomutase-3 locus and is closely linked (within 3.0 centimorgans) to the gene specifying a highly homologous intracellular retinol binding protein known as CRBP. Mouse-human somatic cell hybrids were used to determine that both the CRBP and CRBP II genes are located on human chromosome 3.     

Interactions of retinol with binding proteins: studies with rat cellular retinol-binding protein and with rat retinol-binding protein

The interactions of retinol with rat cellular retinol-binding protein (CRBP) and with rat serum retinol-binding protein (RBP) were studied. The equilibrium dissociation constants of the two retinol-protein complexes (Kd) were found to be 13 x 10(-9) and 20 x 10(-9) M for CRBP and for RBP, respectively. The kinetic parameters governing the interactions of retinol with the two binding proteins were also studied. It was found that although the equilibrium dissociation constants of the two retinol-protein complexes were similar, retinol interacted with CRBP 3-5-fold faster than with RBP; the rate constants for dissociation of retinol from CRBP and from RBP (koff) were 0.57 and 0.18 min-1, respectively. The rate constants for association of retinol with the two proteins (kon) were calculated from the expression: Kd = koff/kon. The kon's for retinol associating with CRBP and with RBP were found to be 4.4 x 10(7) and 0.9 x 10(7) M-1 min-1, respectively. The data suggest that the initial events of uptake of retinol by cells are not rate-limiting for this process and that the rate of uptake is probably determined by the rate of metabolism of this ligand. The data indicate further that the distribution of retinol between RBP in blood and CRBP in cytosol is at equilibrium and that intracellular levels of retinol are regulated by the levels of CRBP.     

Retinoic acid rapidly induces lung cellular retinol-binding protein mRNA levels in retinol deficient rats

Retinol deficiency resulted in decreased mRNA levels for cellular retinol-binding protein (CRBP) in the lungs and the testes. The level of lung CRBP mRNA increased 2.3-fold one hour after oral administration of retinoic acid to retinol deficient rats. In contrast, testicular CRBP mRNA level was not influenced. Our data indicate that retinoic acid regulates CRBP mRNA level in the whole animal and this rapid effect suggests a role for CRBP in the mechanism of vitamin A action at genomic level.     

血清视黄醇结合蛋白4与心血管疾病的研究进展

脂肪组织可分泌大量脂肪细胞因子如脂联素、抵抗素、网膜素、视黄醇结合蛋白4等,这些脂肪细胞因子在心血管疾病发生中的重要作用成为目前的研究热点.在动物与人类的研究中均发现,视黄醇结合蛋白4与胰岛素抵抗、动脉硬化、脂类代谢、高血压等密切相关.本文就相关的进展进行综述.     

Herpes simplex virus binding and entry modulate cell surface protein mobility.

Fluorescence photobleaching recovery measurements showed that herpes simplex virus type 1 attachment to target cells rapidly induced an anchorage modulation of cell surface protein mobility, an activity mediated by the cytoskeleton and associated with the multivalent attachment of other ligands (e.g., cells, lectins, or anti-immunoglobulin) to cell surfaces. The restriction in cell surface protein mobility was released concurrently with virus penetration. The effects of attachment and penetration on cell surface protein mobility and cytoskeletal function are some of the earliest cellular changes induced by herpes simplex virus infection.     

HINT predictive analysis of binding between retinol binding protein and hydrophobic ligands

The interaction between the retinol binding protein and four ligands was evaluated using HINT, a software based on experimental LogP values of individual atoms. A satisfactory correlation was found between the HINT scores and the experimental dissociation constants of three of the ligands, fenretinide, N-ethylretinamide and all-trans retinol, despite their hydrophobic nature. A prediction is made for the binding affinity of the fourth ligand, axerophtene, not yet determined in solution.     

Neonatal estrogenization leads to increased expression of cellular retinol binding protein 2 in the mouse reproductive tract

Exposure to estrogenic substances during a time window, the so-called critical period, in perinatal life causes an irregular development of the genital tract that leads to ovary-independent proliferation and cornification in the vaginal epithelium in mice. We have previously demonstrated that retinol inhibits the irreversible effects of estrogen on the vagina. Here, mice kept in a vitamin-A-deficient condition during perinatal life were shown to be more sensitive to the harmful effects of estrogen. In addition, expression of mRNA for retinol binding protein type 2 (CRBP2), a small intestine-specific cytosolic protein that captures intracellular retinal and retinol, was detected in the vaginal epithelium. Induction of increased expression of CRBP2 mRNA by estrogen was also evident in the uterus and epididymis. Both estradiol-17 and diethylstilbestrol markedly increased the tissue content of CRBP2 mRNA in the vagina and uterus during the neonatal critical period but not after 15 days of age. These results taken together imply that estrogen disrupts the local vitamin A balance by an induction of CRBP2 gene expression in the epithelium in the developing mouse genital tract, and that retinoid imbalance may contribute to the genesis of irreversible effects of estrogen on the vagina.This work was supported by Grants-in-Aid for Priority Area B (to T.M. and M.M.) and for Young Scientists (to M.M.) from the Ministry of Culture, Sports, Science and Technology, Japan