Secretion Of Retinol-binding Protein Research Articles

The effect of estrone and estradiol treatment on endometrial total protein, uteroferrin, and retinol-binding protein secretion during midpregnancy or midpseudopregnancy in swine.

It has been hypothesized that conceptus estrogens influence endometrial protein secretion during pregnancy in swine. To test this hypothesis, the effect of estrone and estradiol treatment from d 30 to 60 of pregnancy or pseudopregnancy on endometrial protein secretion was investigated. Pregnant (P; n = 16) and pseudopregnant (PP) gilts (n = 18) received either sham treatment or estrone or estradiol implants (5 mg/d release rate; 60 d release) on d 30 of P or PP. Blood samples were collected on d 30, 40, 50, and 60 to measure estrone and estradiol. On d 60, gilts were hysterectomized. For P gilts, endometrium in apposition to one placenta from each uterine horn was collected. For PP gilts, each uterine horn was flushed with 40 mL of leucine-deficient minimal essential medium (MEM), and endometrial tissue was collected from each horn. Endometrial tissues were incubated in MEM in the presence of 50 microCi of [3H]leucine to examine protein secretion. Estrone and estradiol treatments increased both plasma and endometrial concentrations of estrone (P < .01 except endometrium for P gilts) and estradiol (P < .01, respectively). Endometrium from P gilts secreted more nondialyzable macromolecules (NDM), acid phosphatase activity (AP, a measure of uteroferrin), and retinol-binding protein (RBP) in culture than did endometrium from PP gilts. Estrone treatment increased (P < .01) endometrial NDM from P gilts but not that from PP gilts; estradiol had no effect. Both estrone and estradiol increased (P = .069) endometrial secretion of AP of PP but not of P gilts. Endometrial secretion of RBP was not affected by either estrone or estradiol treatment. Neither estrone nor estradiol affected total protein or AP and estrone treatment decreased (P < .05) RBP in uterine flushings from PP gilts. These data indicate that endometrium from P pigs secretes more protein than endometrium from PP pigs but neither estrone nor estradiol completely mimics the effect of pregnancy.

N-(4-hydroxyphenyl)retinamide (Fenretinide) and nephrectomy alter normal plasma retinol-binding protein metabolism

We have reported previously that injecting vitamin A-deficient rats with N-(4-hydroxyphenyl)retinamide causes a significant reduction in the liver retinol-binding protein concentration and a 2 fold rise in the kidney retinol-binding protein concentration. This presumably reflects a rapid translocation of retinol-binding protein from the liver to the kidney through the plasma, although no rise in plasma retinol-binding protein is detected. In the present studies, nephrectomized rats were used to determine if retinol-binding protein accumulating in kidneys passes through the plasma. Bilateral nephrectomy in control rats caused the plasma retinol-binding protein concentration to approximately double by 5 hr postsurgery. However, nephrectomy plus N-(4-hydroxyphenyl)retinamide treatment did not result in an increase in the plasma retinol-binding protein concentration. Therefore, the lowering of liver retinol-binding protein concentration in response to N-(4-hydroxyphenyl)retinamide treatment was not accounted for by an accumulation of retinol-binding protein in the plasma compartment. Interestingly, the muscle retinol-binding protein concentration increased with nephrectomy plus N-(4-hydroxyphenyl)retinamide treatment. The ratio of muscle retinol-binding protein:plasma retinol-binding protein in vitamin A-deficient nephrectomized rats treated with N-(4-hydroxyphenyl)retinamide was significantly higher than in comparable rats treated with either carrier or retinol. We conclude that in vivo N-(4-hydroxyphenyl)retinamide induces the secretion of retinol-binding protein from the liver. Since the N-(4-hydroxyphenyl)retinamide-retinol-binding protein complex does not bind with transthyretin it rapidly leaves the plasma. In non-nephrectomized rats this complex is rapidly filtered by the kidney. Nephrectomizing rats causes the retinol-binding protein secreted in response to N-(4-hydroxyphenyl)retinamide to diffuse into interstitial fluid.

Effect of treatment with retinyl palmitate, progesterone, oestradiol and tamoxifen on secretion of a protein similar to retinol-binding protein during uterine gland development in neonatal pigs

Previous work has demonstrated that uterine secretion of a protein with M(r) 20,000 and pI 5.5 increases during neonatal endometrial gland development. Uterine tissue was collected from a 60-day-old gilt and cultured in 0.1 x leucine minimum essential medium (MEM) plus 50 muCi [3H]leucine to determine whether this protein is related to retinol-binding protein (RBP). Conditioned medium was immunoprecipitated using anti-human RBP antiserum. A radioactive protein with M(r) 20,000 and pI 5.5 was specifically immunoprecipitated from the conditioned medium. Uteri from neonatal gilts were collected at birth (day 0) and on days 3, 6, 9 and 12, cultured, and secreted proteins were immunoprecipitated as described above to determine whether secretion of immunoreactive RBP increased coincident with initiation of endometrial gland development. Immunoprecipitation demonstrated that the secretion of immunoreactive RBP increased by day 3. Finally, gilts were treated each day with corn oil, retinyl palmitate (10,000 iu day-1), progesterone (20 mg day-1), oestradiol (100 micrograms day-1) or tamoxifen (1 mg day-1 or 0.1 mg day-1) for 14 days beginning at birth to determine the effects of these treatments on endometrial gland development and uterine secretion of immunoreactive RBP. On day 14, gilts were killed and uteri collected. Uterine tissue samples were prepared for histology (to evaluate uterine development using morphometry) and for culture in 0.1 x methionine MEM plus 25 muCi [35S]methionine (to evaluate uterine protein synthesis).(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the Metabolism of Retinol and Retinol-binding Protein in Transthyretin-deficient Mice Produced by Homologous Recombination

Tissue needs for retinoids are believed to be satisfied through the delivery in the circulation of retinol by its specific plasma transport protein, retinol-binding protein (RBP), which circulates as a 1-to-1 protein complex with transthyretin (TTR). The binding of RBP to TTR is thought to prevent filtration of retinol-RBP in the kidney and to play a role in secretion of RBP from hepatocytes. Recently a strain of mice (TTR-) that totally lacks immunoreactive TTR was produced by targeted mutagenesis. We have explored the effects of TTR deficiency on retinol and RBP metabolism in this mutant strain. In pooled plasma from the TTR- mice retinol levels averaged 6% of those of wild type animals. Similarly, plasma RBP in the TTR- mice was found to be 5% of wild type levels. Hepatic retinol and retinyl ester levels were similar for mutant and wild type mice, suggesting that the mutation affects neither the uptake nor storage of dietary retinol. Levels of retinol and retinyl esters in testis, kidney, spleen, and eye cups from TTR- mice were normal. Plasma all-trans-retinoic acid levels for the TTR- mice were 2.3-fold higher than those of wild type (425 versus 190 ng/dl). Kidney RBP levels were similar for the mutant and wild type mice and we were unable to detect intact RBP in urine from TTR- mice. Hepatic RBP levels in the TTR- mice were 60% higher than those of wild type mice (39.8 versus 25.0 micrograms of RBP/g of tissue). These data may suggest that there is a partial blockage in RBP secretion from TTR- hepatocytes that leads to lessened plasma levels of retinol-RBP.

Synthesis and secretion of retinol-binding protein and transthyretin by cultured retinal pigment epithelium

Recent studies indicate that the retinal pigment epithelium (RPE) may serve as an extrahepatic source of retinol-binding protein (RBP) and transthyretin (TTR) for the retina by virtue of the fact that this cell layer is the exclusive retinal location for mRNA coding for these proteins [Herbert, J., et al. (1991) Invest. Ophthalmol. Vis. Sci. 32, 302-309; Cavallaro, T., et al. (1990) Invest. Ophthalmol. Vis. Sci. 31, 497-501], although the proteins themselves are present in a variety of retinal neurons. It is therefore necessary to determine whether these mRNAs are translated and whether their translated products are secreted like hepatic RBP and TTR. Metabolic labeling of cultured bovine RPE with [35S]cysteine and [35S]methionine and subsequent analysis of newly synthesized proteins in the conditioned medium by affinity chromatography, gel filtration, partial amino acid sequence analysis, and autoradiography of electrophoretograms indicate that both RBP and TTR are synthesized and secreted by the RPE. Moreover, for cells grown in chambers with permeable supports, the predominant direction for secretion was into the apical medium. The mean apical:basal ratio after 72 h of incubation was 9.2 for TTR and 4.5 for RBP. A function for these proteins in the neurosensory retina remains speculative. They could be involved in the delivery of all-trans-retinol to amacrine and Müller cells as a precursor for retinoic acid, since these cells are known to contain cellular retinoic acid binding protein [Gaur, V.P., et al. (1990) Exp. Eye Res. 50, 505-511; Milam et al. (1990) J. Comp. Neurol. 296, 123-129].(ABSTRACT TRUNCATED AT 250 WORDS)

N-(4-Hydroxyphenyl)retinamide (Fenretinide) Induces Retinol-Binding Protein Secretion from Liver and Accumulation in the Kidneys in Rats

The chemopreventive retinoid N-(4-hydroxyphenyl)retinamide (HPR) depresses serum retinol and retinol-binding protein (RBP) concentrations. To study long-term effects of HPR on serum proteins, rats were fed a control diet or a diet containing HPR (737 mumol/kg diet) for 14 d. Serum retinol and RBP of HPR-treated rats decreased to 42 and 41%, respectively, of initial concentrations. Transthyretin, albumin and transferrin did not differ between treatments. Previous studies found that HPR decreased secretion of the retinol-RBP complex into plasma. To investigate acute effects of HPR on RBP metabolism, vitamin A-deficient rats were injected with HPR (51 mumol/kg body wt), retinol (0.52 mumol/rat) or Tween carrier only. Liver RBP concentrations in HPR- and retinol-treated rats were 45 and 18%, respectively, of concentrations in Tween-treated rats, indicating rapid RBP secretion. Tween- and HPR-treated rats maintained relatively constant serum RBP concentrations, whereas retinol-replete rats had 12-fold higher serum RBP after 150 min. Rats treated with HPR and rats treated with retinol had 29- and eightfold higher kidney RBP concentrations, respectively, than Tween-treated rats, indicating rapid clearance of RBP from plasma. We conclude that HPR affects RBP metabolism by inducing secretion of liver RBP into the bloodstream and rapid RBP accumulation in the kidney.

Synthesis and secretion of retinol-binding protein by cultured rat Sertoli cells.

We report here that retinol-binding protein (RBP) is synthesized and secreted by rat Sertoli cells in culture. This was demonstrated in four ways. First, transthyretin (TTR) bound to Sepharose 4B retained a labeled protein from media collected from Sertoli cells provided with 35S-methionine, under the same conditions as authentic RBP is bound. The protein was co-eluted with authentic RBP by pure water. Second, this same radioactive protein co-eluted with pure RBP upon gel filtration. Third, when subjected to SDS-PAGE, the protein again migrated with pure RBP, as shown by radioautography. Finally, Sertoli cells were incubated with 35S-cysteine and the conditioned medium was put over a TTR-Sepharose column to isolate the radioactive protein, as characterized above. Cysteine residues were oxidized to cysteic acid residues, and the protein was submitted for sequencing through the first ten residues. Radioactivity was located only in the fourth residue, where a cysteine residue is found in rat RBP. This indicates that RBP is secreted by the Sertoli cell and may serve as the carrier of retinol to the developing germ cells, which are known to be dependent upon vitamin A.

Differentiation-dependent expression of retinoid-binding proteins in BFC-1 beta adipocytes.

Recently, we demonstrated that adipose tissue plays an important role in retinol storage and retinol-binding protein (RBP) synthesis. Our data suggested that RBP expression in adipose tissue is dependent on the state of adipocyte differentiation. To examine this possibility, we explored the differentiation-dependent expression of RBP using BFC-1 beta preadipocytes, which can be stimulated to undergo adipose differentiation. Total RNA was isolated from undifferentiated (preadipocytes) and differentiated (adipocytes) BFC-1 beta cells and analyzed by Northern blotting. RBP mRNA was not detected in the preadipocytes, but considerable RBP mRNA was present in differentiated BFC-1 beta cells. In BFC-1 beta cells, induced to differentiate with insulin and thyroid hormone, RBP mRNA was first detected after 4 days, reached a maximum level by day 10, and remained at this maximum level for at least 2 more days. Cellular retinol-binding protein was expressed at low levels in the BFC-1 beta preadipocytes and the level of expression increased for 6 days after induction to differentiate and slowly declined on later days. Neither the maximum level of RBP expression nor the day on which this level was reached was influenced by the level of retinol provided in the BFC-1 beta culture medium. BFC-1 beta cells secreted newly synthesized RBP into the culture medium at a rate of 43 +/- 14 ng RBP/24 h/10(6) adipocytes. When the BFC-1 beta adipocytes were provided 1.0 microM retinol in the medium, they accumulated the retinol and synthesized retinyl esters. These studies with BFC-1 beta cells confirm that RBP synthesis and secretion and retinol accumulation are intrinsic properties of differentiated adipocytes. Furthermore, they suggest that RBP and cellular retinol-binding protein gene expression are regulated as part of a package of genes which are modulated during adipocyte differentiation.

Regulation of plasma retinol binding protein secretion in human HepG2 cells

Retinol binding protein (RBP) is the plasma transport protein of retinol. Mobilization of RBP from the liver stores is stimulated by retinol. During vitamin A deficiency, RBP secretion is specifically inhibited while its rate of biosynthesis is unaffected. As a consequence, RBP, as apoprotein, accumulates inside the endoplasmic reticulum (ER) of the hepatocyte, and a new elevated steady-state concentration is reached. We have studied the role of degradation on the regulation of RBP metabolism in retinol deficient HepG2 cells and determined the intracellular site where RBP degradation takes place. Pulse—chase experiments show that RBP half-life is ca.9 h in retinol-depleted cells. RBP degradation is slow and is insensitive to the treatment with NH 4Cl, which inactivates lysosomal proteases and to the drug brefeldin A, which prevents protein export from the ER. The data obtained suggest that RBP degradation occurs, at least in part, in a pre-Golgi compartment. 2-Mercaptoethanol, at millimolar concentration, induces RBP secretion, suggesting a possible role for sulfhydryl-mediated apo-RBP retention by resident ER proteins.

Steroid regulation of the synthesis and secretion of retinol-binding protein by the uterus of the pig

Steroid regulation of the synthesis and secretion of retinol-binding protein by the uterus of the pig

Steroid regulation of the synthesis and secretion of retinol-binding protein by the uterus of the pig.

The endometrium of the pig secretes retinol-binding protein (RBP) under the influence of progesterone (P4). The objective of this study was to determine how conceptus-derived estrogen might modulate this production of RBP around days 11-13 of pregnancy when conceptuses elongate from spheres to long thread-like forms. Concentrations of retinol and RBP were low (35 +/- 7 ng/ml) in uterine flushings obtained on days 10-12 of the estrous cycle or from pregnant gilts in which conceptuses had not elongated. Concentrations of retinol and RBP increased 7- to 8-fold (P less than 0.01) in flushings where filamentous conceptuses were present. Size exclusion and ion exchange chromatography demonstrated that virtually all retinol assayed in uterine flushings was associated with RBP. Northern blot analysis with a cDNA representing uterine RBP revealed a single endometrial mRNA 1.1 kilobases in length. Expression of RBP mRNA in uterine endometrium was measured in ovariectomized prepubertal gilts after the administration of steroids according to the following regimens: I, corn oil (days 0-16; n = 10); II, estradiol benzoate (EB; days 13-14; n = 11); III, EB (days 1-2; n = 12); IV, EB (days 1-2) plus P4 (days 3-16; n = 12); and V, EB (days 1-2) plus P4 (days 3-16) plus EB (days 13-14; n = 12). EB (200 micrograms) and P4 (100 mg) were administered twice daily. Treatment IV was designed to stimulate the estrous cycle, and treatment V simulated early pregnancy. All gilts were hysterectomized on day 16, and total uterine mRNA (3 micrograms) was analyzed by Northern blotting. No RBP mRNA was detected in groups I, II, or III. In group IV, 5 of 12 gilts had detectable RBP mRNA, as measured by densitometric scanning (OD = 0.35 +/- 0.14). RNA isolated from all gilts in group V (12 of 12) gave a strong hybridization signal (OD = 1.58 +/- 0.22) for RBP. Finally, RBP mRNA was examined in the uterine endometrium of mature gilts on day 13 of the estrous cycle (n = 4), day 13 of pseudopregnancy (2.5 mg EB given on days 11-12; n = 4), or day 13 of pregnancy after conceptuses had elongated (n = 4). RBP mRNA was present in all groups, but was enhanced approximately 12-fold (P less than 0.01) in pregnant and pseudopregnant gilts compared to that in control gilts.(ABSTRACT TRUNCATED AT 400 WORDS)

Retinol binding protein is produced by the bovine endometrium and accumulates in uterine secretions in a progesterone-dependent manner

Studies were conducted to establish whether (a) uterine lumenal fluids contain an immunoreactive retinol binding protein (RBP), (b) such a protein is produced by the uterine endometrium, (c) retinol binding activity can be associated with immunoreactive RBP, and (d) presence of the immunoreactive RBP is regulated by progesterone. When using antiserum to human RBP (serum form) as a probe in immunoblotting, three molecular weight classes of protein species could be detected in uterine lavage fluids of pregnant and bred/nonpregnant cows at Day 17 after estrus and in culture supernatants of cyclic, bred/nonpregnant and pregnant cows at Day 17 after estrus. The predominant band was at a molecular weight (Mr) of 22 000 (RBP 22) but several other minor bands could be detected at molecular weights of 25 000–30 000 (RBP 25–30) and 55 000–65 000 (RBP 55–65). These immunoreactive forms of RBP could be detected in serum. Addition of cycloheximide to endometrial cultures inhibited amounts of RBP 22 in supernatants of endometrial cultures, confirming in vitro synthesis of RBP by endometrium. Biological activity was confirmed by incubation of immunoblots with 3H-retinol: specific binding of radiolabel was associated with RBP 22 and RBP 55–65. To investigate regulation of secretion of RBP, ovariectomized cows were treated with corn oil vehicle for 12 days or with 750 mg day −1 progesterone for 12 or 30 days. While immunoblots revealed RBP 22 in uterine lavage fluids of endometrial explants from all cows, progesterone-treated cows had more RBP 22 in uterine lavage fluids than did cows treated with vehicle alone. Staining intensities of RBP 25–35 and RBP 55–65 were not affected by treatment. It was concluded that the uterine lumen contains a retinol binding protein that is derived from both local endometrial synthesis and blood. Furthermore, amounts of RBP in uterine secretions are regulated by progesterone.

Direct mobilization of retinol from hepatic perisinusoidal stellate cells to plasma.

We have studied the mechanism for mobilization of retinol from stellate cells. Our data show that perisinusoidal stellate cells isolated from liver contained retinol-binding protein (RBP) mRNA. By Western blot analysis we found that cultivated liver stellate cells secreted RBP into the medium. Cultivated stellate cells loaded in vitro with [3H]retinyl ester mobilized radioactive retinol as a complex with RBP. Furthermore, exogenous RBP added to the medium of cultured stellate cells increased the secretion of retinol to the medium. These data suggest that liver stellate cells in vivo mobilize retinol directly to the blood and that a transfer to parenchymal cells for secretion as holo-RBP is not required. The direct mobilization of retinol from liver stellate cells as retinol-RBP to blood is indirectly supported by the demonstration of RBP mRNA production and RBP secretion by lung stellate cells. The data suggest that the same mechanism for retinol mobilization may exist in hepatic and extrahepatic stellate cells. This is, vitamin A-storing stellate cells in liver, lungs, and probably also in other organs may synthesize their own RBP (or alternatively use exogenous RBP) and mobilize holo-RBP directly to the blood.

Retinol-binding protein and transthyretin expressed in HeLa cells form a complex in the endoplasmic reticulum in both the absence and the presence of retinol

To establish a suitable experimental system for studies of the interaction of retinol-binding protein (RBP) with transthyretin (TTR) we have expressed the corresponding cDNAs in HeLa cells. To investigate whether complex formation might occur already in the endoplasmic reticulum (ER), the C-terminal ER retention signal, KDEL, was attached to TTR. The tetrameric TTR-KDEL fusion protein was retained in the ER of HeLa cells. When RBP was co-expressed with TTR-KDEL, RBP was retained intracellularly. A cDNA-encoding purpurin, a protein which is 50% identical to RBP, was then expressed together with TTR-KDEL. Purpurin was not retained intracellularly and did not bind to TTR coupled to Sepharose. The effect of the vitamin A status on the secretion of TTR and RBP was examined. While TTR expressed alone was not retained intracellularly, TTR was retained in vitamin A-deficient cells when co-expressed with RBP. Addition of retinol stimulated rapid secretion of both proteins. These results demonstrate that TTR can form a complex with RBP in the ER. The data suggest that RBP and TTR are secreted as a complex.

Ultrastructural localization of plasma retinol-binding protein in rat liver

Immunocytochemical studies were carried out to examine the subcellular localization of plasma retinol-binding protein (RBP) in rat liver. The studies used normal, retinol-deficient, and retinol-repleted retinol-deficient rats with or without colchicine pretreatment. Affinity-purified monomeric Fab' fragments from the IgG fraction of rabbit anti-rat RBP were conjugated to horseradish peroxidase. This conjugate effectively penetrated into tissue sections and enabled RBP to be localized by high resolution immunoelectron microscopy. In the normal liver parenchymal cell, RBP was found to be localized in the synthetic and secretory structures including endoplasmic reticulum (ER), Golgi complex (GC), and secretory vesicles. With the method used, significant localization of RBP was not observed in hepatic cells other than parenchymal cells. The distribution of RBP-positive areas within parenchymal cells changed markedly with retinol depletion. Thus, a heavy accumulation of RBP in the ER, accompanied by a marked decrease of the RBP-positive GC and secretory vesicles, was demonstrated in liver parenchymal cells from retinol-deficient rats. After repletion of deficient rats with retinol, the RBP that accumulated in the ER appeared to move rapidly from the ER through GC and secretory vesicles to the cell surface. Pretreatment with colchicine led to marked increase in RBP-positive secretory vesicles in retinol-repleted rat liver parenchymal cells. The results reported here demonstrate that the specific block in hepatic RBP secretion seen in retinol deficiency involves an inhibition of the movement of RBP from the ER to the GC in the parenchymal cell.

Regulation of metabolism of retinol-binding protein by vitamin A status in children with biliary atresia

Regulation of retinol-binding protein (RBP) by vitamin A status was studied in 43 children; 25 had biliary atresia and vitamin A deficiency, 15 had biliary atresia treated by vitamin A, and 9 control children had normal liver and vitamin A status. Vitamin A and RBP were assayed and the two forms of RBP, holo-RBP and apo-RBP, were separated in both liver and plasma. No difference in liver RBP concentrations was found between the three groups; apo-RBP was the most abundant form. Plasma RBP concentrations and the ratio of retinol to RBP were lower for vitamin A-deficient than for vitamin A-treated children. Two models could be proposed: 1) a preferential secretion of holo-RBP with variations in RBP catabolism or synthesis in vitamin A-deficient liver and 2) a continuous secretion of RBP by the liver with a rapid clearance of plasma apo-RBP in vitamin A deficiency.

Secretion pattern of retinol-binding protein in blood of goats: effect of vitamin A, provitamin A and their dosing schedule.

Studies were conducted on the secretion of retinol-binding protein (RBP) in blood of goats given different treatments of preformed vitamin A, beta-carotene and plant carotenoids. Administration of these sources either in a single massive dose or massive dose split into four equal doses, markedly increased the secretion of RBP in blood. The secretion of RBP in blood occurred at least in two phases, one at early periods and the other at later periods.

Induction of the expression of retinol-binding protein and transthyretin in F9 embryonal carcinoma cells differentiated to embryoid bodies.

Studies were conducted to determine if the expression of the gene for retinol-binding protein (RBP) and/or transthyretin (TTR) could be induced upon differentiation of F9 teratocarcinoma cells to either visceral endoderm or parietal endoderm. Both TTR mRNA and RBP mRNA were undetectable in the undifferentiated F9 stem cells and in F9 cells differentiated to parietal endoderm. However, TTR mRNA and RBP mRNA were both detected in F9 cell aggregates differentiated to embryoid bodies (which contain visceral endoderm-like cells) by treatment of the aggregates in suspension with retinoic acid. TTR mRNA was observed at 3 days, and RBP mRNA at 5 days, after treatment of the F9 cell aggregates with retinoic acid. Both TTR mRNA and RBP mRNA were found to be specifically localized by in situ hybridization in the outer layer of cells (the visceral endoderm-like cells) of the embryoid bodies. Finally, synthesis and secretion of both RBP and TTR by F9 cell embryoid bodies was demonstrated by specific immunoprecipitation of each newly synthesized protein from the culture medium. These data thus demonstrate the production and presence of RBP mRNA and TTR mRNA, and the synthesis and secretion of RBP and TTR, by F9 cell embryoid bodies (specifically by visceral endoderm-like cells). This finding suggests that these two proteins may be synthesized by rodent embryos extremely early in embryonic development.

Retinol-binding protein synthesis and secretion by the rat visceral yolk sac. Effect of retinol status.

Studies were conducted to explore in rats the role of retinol in the regulation of the synthesis and secretion of retinol-binding protein (RBP) by the visceral yolk sac compared to the liver. Previous studies have shown that in retinol deficiency, hepatic RBP secretion is specifically inhibited, whereas hepatic RBP synthesis rate is unchanged. Retinol-depleted, retinoic acid-supplemented female rats were mated, and maternal liver, fetal liver, and visceral yolk sac were obtained at 14 days of gestation (retinol-depleted group). A group of identically treated, retinol-depleted rats were repleted with retinol on the 14th day of gestation, and the same tissues were collected 6 h later (retinol-repleted group). Normal female rats were used as controls. RBP was assayed by radioimmunoassay and RBP mRNA levels by RNase protection assay using a rat RBP cDNA clone. RBP levels in the visceral yolk sac were elevated 10-fold in the retinol-depleted as compared to the control rats and had declined to near normal values in the retinol-repleted animals. The relative levels of RBP mRNA in the visceral yolk sac were very similar in all three groups of rats. Thus, as in the liver, in the visceral yolk sac retinol deficiency inhibits RBP secretion without altering RBP mRNA levels. In the visceral yolk sac, as in the liver, retinol status appears to regulate RBP secretion specifically, without affecting the rate of RBP biosynthesis.

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.