The Journal of Nutrition Vol. 127 No. 1 January 1997, pp. 13-17
Copyright ©1997 by the American Society for Nutritional Sciences

Retinoic Acid Increases Cellular Retinol Binding Protein II mRNA and Retinol Uptake in the Human Intestinal Caco-2 Cell Line^1,2

Marc S. Levin³ and Alan E. Davis

Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

ACKNOWLEDGMENTS

LITERATURE CITED

ABSTRACT

Cellular retinol binding protein II (CRBPII) is an abundant small intestinal protein that facilitates vitamin A trafficking and metabolism. The magnitude of retinol uptake and metabolism correlate to CRBPII levels in the human intestinal Caco-2 cell line. To investigate the importance of retinoic acid receptor response elements in the promoter of the CRBPII gene, retinoic acid regulation of CRBPII expression and vitamin A absorption was studied in differentiated Caco-2 cells. All-trans- or 9-cis-retinoic acid increased CRBPII mRNA levels two- to threefold. This was associated with a 50% increase in retinol absorption. Retinoic acid receptor  and apolipoprotein A1 regulatory protein-1, two nuclear receptors that bind to the CRBPII promoter, were also induced, whereas other retinoid and orphan receptors were not. Thus, retinoic acid may regulate CRBPII expression directly or by selectively changing levels of nuclear receptors or other factors. These studies are the first to demonstrate that retinoic acid can modulate endogenous CRBPII mRNA levels and retinol absorption in Caco-2 cells and suggest that human intestinal vitamin A absorption may be regulated by retinoids.

INTRODUCTION

Vitamin A is an essential nutrient required for normal development and growth. Small intestinal enterocytes play a central role in the assimilation and metabolism of dietary retinol and pro-vitamin A carotenoids [reviewed in Levin (1994)]. Cellular retinol binding protein II (CRBP II)⁴ is an abundant cytosolic protein found predominantly in small intestinal enterocytes (Levin et al. 1987, Li et al. 1986, Ong 1984). CRBP II appears to function to facilitate intestinal vitamin A trafficking and metabolism [reviewed in Levin (1994) and Ong et al. (1994)]. For example, using Caco-2 human intestinal cell lines stably transfected with CRBP II, we have demonstrated that the magnitude of retinol uptake, esterification with long-chain fatty acids and secretion are modulated by CRBP II (Levin 1993, Lissoos et al. 1995). In addition, CRBP II bound retinol appears to be the principle substrate for intestinal lecithin retinol acyltransferase, which catalyzes the transfer of long-chain fatty acids from lecithin to retinol (Ong et al. 1987).

Although dietary administration of retinyl acetate to rats or of 9-cis-retinoic acid to mice did not alter jejunal CRBP II levels (Allegretto et al. 1995, Suzuki et al. 1995), CRBP II levels have been shown to be altered in several physiologic settings which may be associated with the need to increase the efficiency of intestinal retinol absorption. For example, intestinal levels of CRBP II increase dramatically in the peripartum period and in lactating rat dams (Levin et al. 1987, Quick and Ong 1989). CRBP II expression is also markedly induced in the rat remnant small intestine, which undergoes an adaptive response following massive small bowel resection (Dodson et al. 1996, Wang et al. 1995). In addition, rat intestinal CRBP II levels may be increased by vitamin A deficiency (Rajan et al. 1990). Thus, these data suggest that dietary vitamin A assimilation could be regulated by changes in intestinal CRBP II levels.

The mechanisms underlying the physiologic changes in CRBP II expression have not been studied. The human CRBP II gene has not been analyzed; however, the promoters of the murine and rat CRBP II genes contain retinoic acid receptor response elements (RARE) (Mangelsdorf et al. 1991, Nakshatri and Chambon 1994). The murine CRBP II gene possesses at least three RARE designated as RE1, RE2 and RE3 (Nakshatri and Chambon 1994). The latter two are conserved in the rat CRBP II gene, which also contains a retinoid X receptor response element (RXRE). These response elements bind to homodimeric and heterodimeric complexes of ligand bound-retinoic acid receptors (RAR) and retinoid X receptors (RXR) in addition to homodimeric and heterodimeric complexes of orphan nuclear receptors such as apoA1 regulatory protein (ARP-1), hepatocyte nuclear receptors (HNF-4) and v-erbA related protein 3 (EAR-3) (COUP-TF) (Nakshatri and Chambon 1994).

Because retinoic acid can directly modulate gene expression and is synthesized and metabolized in the small intestine [reviewed in Levin (1994)], the goal of this study was to better define the role of retinoic acid in regulating intestinal CRBP II expression and vitamin A absorption in intact cells. The human intestinal cell line, Caco-2, was used for these studies because Caco-2 cells form polarized monolayers possessing many features of the mature small intestinal mucosa and because they express CRBP II and are able to absorb, esterify and secrete retinol (Levin 1993, Lissoos et al. 1995, Quick and Ong 1990). In addition, as shown below, Caco-2 cells express the nuclear retinoic acid receptors RAR, RAR, RAR and RXR, in addition to the orphan receptors HNF-4, ARP-1 and EAR-3.

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MATERIALS AND METHODS

Materials. Fetal calf serum was from JRH Biosciences (Lenexa, KS) and ITS premix from Collaborative Biomedical Products (Bedford, MA). [11,12-³H(N)]Retinol (specific activity 1.5-2.2 TBq/mmol) was obtained from Amersham (Arlington Heights, IL); all-trans-retinol and retinoic acid were from Sigma (St. Louis, MO); 9-cis-retinoic acid from Hoffman LaRoche (Nutley, NJ) and all high performance liquid chromatography solvents were from Fisher Scientific (Springfield, NJ). All retinoid-containing solutions were purged with N₂ and were handled on ice in red light (60 W) and/or dim lighting. The purity of retinoid stocks was determined prior to use by absorption spectrophotometry and high performance liquid chromatography as described (Levin 1993, Lissoos et al. 1995).

Cell culture. Caco-2 cells from the American Type Culture Collection were maintained as previously described at 37°C, 5.5% CO₂ in Dulbecco's modified Eagle's medium containing 6 mmol/L glutamine, 0.1 mmol/L nonessential amino acids, 44 mmol/L NaHCO₃, 1 × 10⁵ units/L penicillin, 100 g/L streptomycin and 10% heat-inactivated fetal calf serum (Levin 1993, Lissoos et al. 1995). Cells were plated at 1.2 × 10⁴ cells/cm² in uncoated plastic flasks.

Effects of retinoic acid administration. Differentiated Caco-2 monolayers (14-18 d postconfluent) in T175 cm² flasks were used. For experimental studies, serum was removed by washing with 50 mL of Dulbecco's phosphate buffered saline (PBS). Cells were pre-incubated for 2 h with serum-free medium containing 6 mmol/L Na-taurocholate and 0.05 µmol/L retinol supplemented with ITS premix (final concentration insulin 5 µg/mL, transferrin 5 µg/ml, selenious acid 38.8 nmol/L). After incubation for 2 h at 37°C, the medium was replaced with fresh media containing all-trans-retinoic acid (0.2, 1 or 5 µmol/L), 9-cis-retinoic acid (5 µmol/L) or vehicle alone (ethanol, final concentration <0.022 mol/L). Cell monolayers were incubated an additional 24 or 72 h and rinsed three times with ice-cold PBS; then half of each flask was harvested for isolation of RNA and half for analysis of retinol absorption as described below. Base-line data were also obtained from flasks that were harvested immediately after the change to fresh medium containing the vehicle alone.

Northern blot analysis. For CRBP II mRNA analysis, cells were scraped in guanidine thiocyanate (4 mol/L), sodium citrate (25 mmol/L) and n-lauroyl sarcosine (17 mmol/L). RNA was isolated and Northern blots were prepared, probed with radiolabeled cDNAs and then washed as described (Levin 1993, Levin et al. 1987). Northern blots were serially hybridized with the following cDNA probes: rat CRBP II cDNA (Li et al. 1986), human -actin [(Gunning et al. 1983), to normalize for loading and transfer efficiency], human apo A-1 (Roghani and Zannis 1988), murine cellular retinoic acid binding protein (CRABP I, Boylan and Gudas 1991), human RXR (Mangelsdorf et al. 1990), human RAR (Giguere et al. 1987, Petkovich et al. 1987), human RAR (Benbrook et al. 1988, Brand et al. 1988), human RAR (Krust et al. 1989), rat HNF-4 (Ladias et al. 1992, Sladek et al. 1990), human ARP-1 (Ladias and Karathanasis 1991) and human EAR-3 (Ladias et al. 1992). Prior to being reprobed, Northern blots were stripped and removal of radioactivity confirmed by autoradiography (Levin 1993, Levin et al. 1987). For quantification, autoradiograms were scanned with an Ultroscan XL laser densitometer (Pharmacia LKB Biotechnology, Milwaukee, WI) or with a UMAX Powerlook high resolution scanner used in conjunction with Adobe Photoshop 2.5 and NIH Image 1.54. 

Analysis of retinol absorption. Analysis of retinol absorption was done as described in Levin (1993). Monolayers were incubated with 5 nmol/L [11,12-³H(N)]retinol (final retinol specific activity ~150 GBq/mmol) for the final 4 h of the incubation period. Cells were scraped in 5 mL of protease inhibition buffer [2 mmol/L Tris HCl (pH 7.1), 50 mmol/L mannitol, 1.54 µmol/L aprotinin, 1 mmol/L phenylmethanesulfonyl fluoride and 1.46 mmol/L pepstatin] and pelleted by centrifugation at 1200 rpm. Cell pellets were resuspended in 0.5 mL protease inhibition buffer and lysates were prepared by sonication. The amount of cell-associated radiolabel was quantified by liquid scintillation spectrometry. Retinol uptake was corrected for recovery of radiolabel and expressed per milligram of cell protein. Cell-associated radiolabel reliably mirrors retinol absorption because secretion of radiolabeled retinoids is minimal after a 4-h pulse under these conditions (Levin 1993).

Statistical methods. All studies were performed with n  3. Statistical significance was accepted at the P  0.05 level. Statistical analyses performed with Excel Release 4.0 (Microsoft, Redmond, WA) include ANOVA and Student's t test as indicated.

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RESULTS

Effects of retinoic acid on CRBP II expression and retinol absorption. To study the effects of retinoic acid administration on CRBP II expression, differentiated Caco-2 monolayers were pulsed with 5µmol/L all-trans-retinoic acid or vehicle alone for 24 or 72 h or with 5µmol/L 9-cis-retinoic acid for 24 h. This dosage is well within the dose range used in cell culture studies of transcriptional regulation and of cell differentiation by retinoic acid. Furthermore, 0.2 and 1 µmol/L doses of all-trans-retinoic acid also induced CRBP II mRNA levels (magnitude of induction ~50-75% of that seen with 5 µmol/L dose; data not shown). To eliminate potential confounding effects from retinol deprivation as a result of fetal calf serum removal, all experiments were done in the presence of 0.05 µmol/L retinol, which is equivalent to the amount present in the maintenance media (Levin 1993). Nevertheless, the same results were obtained in pilot studies done in the absence of added retinol. There were no detectable changes in Caco-2 morphology induced by retinoic acid in our studies.

Compared with the vehicle controls, at 24h, CRBP II mRNA was induced 3.7-fold (P < 0.00003) by all-trans-retinoic acid and 2.3-fold (P < 0.002) by 9-cis-retinoic acid (Fig. 1A and B). At 72 h, CRBP II mRNA was still significantly induced (>2.2-fold; P < 0.0005). Incubation with the ethanol vehicle alone had no effect on CRBP II expression after correction for -actin expression.

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Fig. 1. Effect of all trans- and 9-cis-retinoic acid on CRBP II mRNA levels and retinol absorption by Caco-2 cells. Panel A: Representative autoradiogram of a Northern blot hybridized to a radiolabeled rat CRBP II cDNA followed by stripping and rehybridization with a radiolabeled human -actin cDNA. RNA was extracted from 14-d postconfluent differentiated Caco-2 monolayers treated with vehicle alone (ethanol, final concentration <0.022 mol/L) for 0 h (first 3 lanes only) or 24 h; or with 5 µmol/L all-trans-retinoic acid (at-RA) or 5µmol/L 9-cis-retinoic acid (9C-RA) for 24 h. Cells were incubated in serum-free medium containing 6 mmol/L Na-taurocholate and 0.05 µmol/L retinol supplemented with ITS premix (final concentration insulin 5 mg/L, transferrin 5 mg/L, selenious acid 38.8 nmol/L) as described in Materials and Methods. Panel B: Caco-2 monolayers were treated for 24 or 72 h with vehicle (n = 7 and 3, respectively), all-trans-retinoic acid (n = 7 and 3) or 9-cis-retinoic acid (n = 4; 24 h only); Northern blots were prepared and hybridized as described in Panel A and Materials and Methods. To quantitate CRBP II mRNA levels, autoradiograms were scanned with a UMAX Powerlook high resolution scanner used in conjunction with Adobe Photoshop 2.5 and NIH Image 1.54. All data were normalized to -actin and are expressed in arbitrary densitometric units as mean values ± SEM. *P < 0.002 compared with vehicle control. Panel C: Retinol absorption by Caco-2 monolayers treated with all-trans-retinoic acid or vehicle alone was determined as described in Materials and Methods. In brief, monolayers were incubated with 5 nmol/L [11,12-³H(N)]retinol (final retinol specific activity ~150 GBq/mmol) for the final 4 h of the incubation period. Flasks were washed with cold phosphate buffered saline and the cells were then scraped, pelleted and sonicated. Cell associated radiolabel was quantified by liquid scintillation spectrometry. Data shown are mean ± SEM (pmol retinol/mg total cellular protein) and are normalized for recovery of radiolabel and total cellular protein. *P < 0.04 compared with vehicle control; #P < 0.004 compared with 24-h retinoic acid data. (n  4 at 24 h, n = 3 at 72 h).
[View Larger Version of this Image (42K GIF file)]

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The magnitude of retinol absorption was determined to assess whether induction of CRBP II mRNA levels could be linked to changes in retinol trafficking (Fig. 1C). In independent experiments, induction of CRBP II mRNA by retinoic acid was associated with significant increases in retinol uptake at 24 h (1.3- to 1.6-fold, P < 0.01). Administration of 9-cis-retinoic acid for 24 h resulted in an equivalent stimulation of retinol absorption (1.3-fold, P < 0.04). After 72 h, retinol uptake was increased 1.5-fold (P = 0.002) by all-trans-retinoic acid compared with the vehicle and 1.3-fold (P < 0.004) compared with retinoic acid treatment for 24 h.

Specificity of the induction of CRBP II by retinoic acid. To address the specificity of the induction of CRBP II by retinoic acid, the expression of an additional retinoic acid-responsive gene, apoA1 and of another gene involved in retinoid metabolism, cellular retinoic acid binding protein I, were determined. ApoA1 contains a DR-2 RARE element and a DR-1 RXRE. The latter element, which can also bind ARP-1 and HNF4, is closely related to the RXRE found in the rat CRBP II gene. Despite the presence of these response elements and the observed retinoid responsiveness of apoA1 in vitro in HepG2 cells (Rottman et al. 1991), in vivo in the liver of vitamin A-deficient rats and in one report in the rat intestine (though the actual intestinal segment analyzed was not specified; Nagasaki et al. 1994), this gene was not induced by all-trans- or 9-cis-retinoic acid in Caco-2 cells (Fig. 2). Although the precise function of CRABP I has not been determined, it is thought to mediate the metabolism of retinoic acid and may affect the transport of retinoic acid and isomers such as 9-cis-retinoic acid to the cell nucleus [reviewed in Ong et al. (1994)]. As can be seen in Figure 2, CRABP I was not regulated by all-trans- or 9-cis-retinoic acid in these experiments. Thus neither apoAI nor CRABP I was induced by retinoic acid in Caco-2 cells. The inability of retinoic acid to induce these genes suggests that the induction of CRBP II is not simply a phenotypic marker of a more generalized response to retinoic acid administration.

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Fig. 2. All trans- and 9-cis-retinoic acid do not affect apo A-I or cellular retinoic acid binding protein (CRABP) mRNA levels in Caco-2 cells. Panel A: Representative autoradiograms of a Northern blot hybridized sequentially with radiolabeled human apo A-I (apo A-I), cellular retinoic acid binding protein (CRABP) and -actin cDNAs. Before hybridizing with CRABP and -actin cDNAs, the blot was stripped and re-exposed to film to confirm the adequacy of stripping. RNA was extracted from 14-d postconfluent differentiated Caco-2 monolayers treated with vehicle alone (ethanol, final concentration <0.022 mol/L) for 0 h (first 3 lanes only) or 24 h; or with 5µmol/L all-trans-retinoic acid (at-RA) or 5 µmol/L 9-cis-retinoic acid (9C-RA) for 24 h. Cells were incubated in serum-free medium containing 6 mmol/L Na-taurocholate and 0.05 µmol/L retinol supplemented with ITS premix (final concentration insulin 5mg/L, transferrin 5 mg/L, selenious acid 38.8 nmol/L) as described in Materials and Methods. Panel B: To quantitate apo A-I and CRABP II mRNA levels, autoradiograms were scanned with a UMAX Powerlook high resolution scanner used in conjunction with Adobe Photoshop 2.5 and NIH Image 1.54. All data were normalized to -actin and are expressed in arbitrary densitometric units as mean values ± SEM (n = 7 for vehicle, n = 4 for apo A-I and for CRABP).
[View Larger Version of this Image (56K GIF file)]

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Expression of retinoid and orphan nuclear receptors in retinoic acid-treated Caco-2 cells. To begin to characterize the mechanism by which retinoic acid induced CRBP II in Caco-2 cells, the expression and retinoic acid responsiveness of several transcription factors that can bind to putative receptor binding elements in the promoter of the CRBP II gene were examined. As demonstrated in Fig. 3, Northern blots were probed with radiolabeled cDNA probes for RXR, RAR, RAR, RAR, and the orphan receptors HNF-4, ARP-1 and EAR-3. By 24 h, retinoic acid induced the expression of RAR, which contains a RARE, by 3.6-fold (P < 0.007, n = 4 pairs normalized to -actin, paired one-tailed Student's t test) and that of ARP-1 by 1.6-fold (P = 0.043, n = 5 pairs). The expression of the other receptors was unaffected.

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Fig. 3. Retinoic acid increases RAR and ARP-1 mRNAs in Caco-2 cells. Caco-2 monolayers were treated for 24 h with vehicle (V) or 5 µmol/L all-trans-retinoic acid (R) (see Fig. 1 and Materials and Methods). Panel A: Northern blots prepared and hybridized as described in Figure 2 were probed with radiolabeled cDNAs for the indicated nuclear retinoid (RXR, RAR, RAR, RAR) and orphan (HNF-4, ARP-1, EAR-3) receptors. The bands labeled with arrows were used for quantitation by densitometry. Panel B: To quantitate mRNA levels, autoradiograms were scanned with an Ultroscan XL laser densitometer. The data shown are arbitrary densitometric units (mean ± SEM) after normalization with human -actin. *P < 0.05 compared with vehicle control.
[View Larger Versions of these Images (48 + 17K GIF file)]

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DISCUSSION

The detection of retinoic acid response elements in the CRBP II promoter and evidence that CRBP II levels are physiologically regulated provided the rationale for determining if retinoids could directly modulate endogenous CRBP II expression and intestinal vitamin A trafficking. Our data indicate that all-trans- and 9-cis-retinoic acid can regulate intestinal CRBP II mRNA accumulation and retinol absorption. A transcriptional mechanism is likely, though these data are certainly consistent with post-transcriptional mechanisms. Regardless, these data indicate that intestinal vitamin A absorption may be subjected to autoregulation by dietary and/or plasma retinoids. In this context, it is interesting that retinoic acid administration also increases the activity of rat hepatic lecithin:retinol acyltransferase activity, which promotes hepatic storage of retinyl esters (Matsuura and Ross 1993).

Mangelsdorf et al. (1991) demonstrated that the rat CRBP II promoter could drive expression of the chloramphenicol acetyl transferase (CAT) gene in the presence of 9-cis- or all-trans-retinoic acid. This effect was mediated by RXR homodimers (formed in the presence of 9-cis-retinoic acid) and could be inhibited by RXR-RAR heterodimers. Thus, reduced levels of 9-cis-retinoic acid or increased levels of RAR might repress rat CRBP II expression. These data imply that CRBP II expression may be regulated in vivo by differential changes in the composition and concentration of intracellular retinoids and retinoic acid receptors. It should be noted, however, that the murine CRBP II promoter was not regulated by retinoic acid in transiently transfected Caco-2 cells (Nakshatri and Chambon 1994). By contrast, our data suggest the endogenous human CRBP II promoter may be regulated by retinoic acid in Caco-2 cells. It should be emphasized that our studies were done to study expression of the endogenous CRBP II gene using postconfluent differentiated cells, which express higher levels of CRBP II than less differentiated cells (Levin 1993). Thus, the apparent inability of retinoic acid to stimulate murine CRBP II expression in transfected Caco-2 cells may be attributed to the use of undifferentiated, freshly plated Caco-2 cells, to differences in the murine CRBP II promoter or to the absence of important cis-acting elements in the promoter constructs used in the transfection studies (Nakshatri and Chambon 1994).

Based on the ability of RXR and RAR to form dimeric complexes, we examined the effect of retinoic acid administration on the expression of RXR, RAR, RAR, RAR, and the orphan nuclear receptors HNF-4 and ARP-1. Steady-state levels of RAR and ARP-1 mRNA were increased by retinoic acid. RXR can form heterodimers in vitro with each of these receptors, and these heterodimers can repress expression driven by the rat CRBP II RXRE (Mangelsdorf et al. 1991, Widom et al. 1992). The observation that induction of CRBP II mRNA accumulation occurred concomitantly with induction of RAR and ARP-1 could be explained in several different ways: 1) receptor mRNA levels may not directly correlate with cellular receptor levels because of post-transcriptional events; 2) receptor levels attained may be insufficient to inhibit CRBP II expression in intact intestinal cells; 3) dimeric complexes containing ARP-1 or RAR may not be inhibitory to the endogenous human CRBP II gene in intact cells; 4) receptor induction may be part of an autoregulatory feedback loop modulating the magnitude and/or duration of CRBP II induction; and 5) other unidentified factors may be more important regulators of the CRBP II promoter. Thus, although one cannot infer from these data that induction of RAR or ARP-1 directly affects the expression of CRBP II, they do indicate that retinoic acid, in addition to directly inducing changes in gene expression, may indirectly regulate CRBP II and other genes by inducing changes in the cellular composition (and occupancy) of retinoid and orphan receptors. The observation that jejunal CRBP II levels were increased in rats fed a diet rich in long-chain triglycerides (Goda et al. 1994, Suruga et al. 1995) suggests that interactions between RXR and peroxisome proliferator-activated receptor(s) may be one such mechanism for modulating CRBP II expression. To further analyze the interactions of the various nuclear receptors with the human CRBP II promoter will require analysis of the human CRBP II gene.

In summary these studies demonstrate that retinoic acid stimulates the accumulation of CRBP II mRNA and retinol absorption in intact intestinal cells. Evidence that this is a specific response can be inferred from the inability of retinoic acid to affect the expression of apoA1 or CRABP. These studies also indicate that retinoic acid can stimulate the expression of RAR and the orphan receptor ARP-1 in Caco-2 cells. Thus, these studies are the first to demonstrate that retinoic acid can directly modulate intestinal CRBP II mRNA levels and vitamin A absorption, thus suggesting that vitamin A absorption may be subjected to autoregulation by dietary and plasma retinoids.

FOOTNOTES

Manuscript received 4 June 1996. Initial reviews completed 29 July 1996. Revision accepted 30 August 1996.

ACKNOWLEDGMENTS

We gratefully acknowledge the assistance of Elzbieta Swietlicki. The following providers of cDNA clones are also gratefully acknowledged: Joseph Grippo (Hoffman LaRoche, Nutley, NJ) for RXR, RAR, and RAR; Lorraine Gudas (Cornell University, Ithaca, NY) for CRABP I; and John Ladias (Harvard University, Cambridge, MA) for HNF-4, ARP-1 and EAR-3.

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