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Originally published In Press as doi:10.1074/jbc.M302551200 on July 3, 2003

J. Biol. Chem., Vol. 278, Issue 37, 35819-35825, September 12, 2003
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Cellular Retinoic Acid-binding Protein II Gene Expression Is Directly Induced by Estrogen, but Not Retinoic Acid, in Rat Uterus*

Xiao-Hong Li and David E. Ong {ddagger}

From the Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232

Received for publication, March 12, 2003 , and in revised form, June 2, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been suggested that cellular retinoic acid-binding protein (II) (CRABP(II)) may have a role in the movement of retinoic acid (RA) to its nuclear receptors, thereby enhancing the action of RA in the cells in which it is expressed. RA has also been shown to increase expression of CRABP(II). Previous work from our laboratory has shown that 17{beta}-estradiol (E2) administration to prepubertal female rats leads to acquisition of the ability of the lining epithelium to synthesize RA as well as to express CRABP(II). To determine whether this appearance of CRABP(II) was dependent on the production of RA, both E2 and RA were administered to ovariectomized rats. E2 administration induced expression of the CRABP(II) gene in the uterus within 4 h, and this induction was not inhibited by prior administration of puromycin, indicating that the induction was direct. In contrast, RA caused no change in CRABP(II) message level, even at times as late as 48 h after administration. Isolation and analysis of 4.5 kb of the 5'-flanking region of the gene revealed no apparent E2-response element. Using this portion of the gene to drive expression of the luciferase gene in transfected cells allowed identification of a region containing an imperfect estrogen-response element and estrogen-response element half-site, necessary for E2-driven induction. A possible Sp1 binding site in the 5'-flanking region of the CRABP(II) gene was also required for this induction. The ability of E2 to induce expression of CRABP(II) suggests that it can enhance the activity of RA, directly affecting expression of retinoid-responsive genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The vitamin A metabolite all-trans-retinoic acid (RA)1 regulates multiple biological processes, including cell proliferation and differentiation, by virtue of its ability to modulate the rate of transcription of numerous target genes. The transcription activities of this hormone are mediated by a family of proteins, the retinoic acid receptors (RAR{alpha}, {beta}, and {gamma} and isoforms). These receptors bind to specific response elements in the promoter regions of target genes and work as ligand-inducible transcription enhancers and repressors (1).

The numerous sites of action of retinoic acid, e.g. from reproductive organs to the respiratory system, suggest that there will be several different factors that regulate its synthesis. One such factor may be estrogen. Administration of E2 to the prepubertal rat leads to a gain of the ability of the uterine lining epithelial cells to synthesize RA (2). Coincident with this gain of ability is the appearance of CRABP(II) within these cells. CRABP(II) is a member of a large family of small proteins that specifically bind lipophilic compounds such as fatty acids and retinoids (3).

Recent work has suggested that CRABP(II) may have a role in the movement of RA to the RARs, thereby enhancing the action of RA in the cells in which it is expressed (4–7). Consistent with that idea, we have now observed coincident appearance of the ability to synthesize RA and expression of CRABP(II) in certain cell types in addition to the uterine epithelium: the developing granulosa cells of the ovarian follicle prior to ovulation and in stromal cells of the uterus undergoing the process of decidualization (2, 8, 9). Human mammary ductal epithelium also has the ability to synthesize RA, and those cells express CRABP(II) as well (10).

Increased expression of CRABP(II) has been noted after administration of RA to intact skin or to cells in culture (11, 12). Analyses of the promoter regions of the human and murine genes have revealed the presence of RA-response elements (RAREs) (13, 14). However, no prototypical estrogen-response element (ERE) was observed for the promoter region for the CRABP(II) gene of these two species. This suggests that the induction of expression of the native rat CRABP(II) gene that we have observed after E2 administration may require the initiation of RA biosynthesis.

To address this question, we have examined expression of CRABP(II) after administration of either E2 or RA to the ovariectomized rat. Only E2 induced expression of CRABP(II). Further analysis of the rat CRABP(II) promoter region demonstrated that the E2 is acting through both an imperfect ERE and a possible Sp1-response element.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Collection—Female ovariectomized Sprague-Dawley rats (180–200 g) were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN). Rats were housed in a temperature- and light-controlled room (±21 °C; lights on 0700–1900 h), fed rat chow (Ralston Purina Co., St. Louis, MO), provided with water ad libitum, and allowed to acclimate for 2 weeks before use. Rats were divided into six groups, each with at least three animals, and injected intraperitoneally with corn oil, puromycin (10 mg/rat), E2 (10 µg/rat), puromycin plus E2, RA (500 µg/rat), or puromycin plus RA, respectively. Puromycin was injected 30 min before injection with E2 or RA, for those experimental groups. After 4 h, uteri were harvested from the animals for RNA extraction. These studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with the oversight of veterinarian of our local institutional animal care and use committee.

RNA Extraction and RNase Protection Assays (RPA)—Total RNA was extracted from individual rat uterus using TRIzol reagent (Invitrogen) and quantified by spectrophotometry. The antisense riboprobe for rat CRABP(II) and cyclophilin was transcribed using the MAXIscript kit (Ambion Inc., Austin, TX) and [{alpha}-32P]UTP (10 Ci/ml; PerkinElmer Life Sciences). The RPAs were carried out using the RPA III kit (Ambion Inc., Austin, TX) according to the user's manual. Briefly, samples of total RNA were hybridized for 15–18 h at 50 °C with excess radiolabeled antisense riboprobe (n = 3 individual samples/time point) and digested by RNase at 37 °C for 30 min. The hybridized products were submitted to electrophoresis on 6% acrylamide gels containing 8% urea. Gels were exposed to BioMAX MR film (Eastman Kodak Co.) with intensifying screens for up to 3 days. Loading variation between samples was standardized by including cyclophilin riboprobe in all hybridization reactions.

Cloning, Southern Blotting, and Preparation of DNA Constructs—A portion of the rat CRABP(II) gene was cloned from a P1 phage library (Incyte Genomics Inc., Palo Alto, CA). The library was screened using PCR primers that amplified a portion of exon 1. The positions of the primers were: 5' primer, +4 to +84, and 3' primer, +162 to +182, numbered according to the transcription start site. One positive clone was identified. It was digested with EcoRI or HindIII, and subsequent Southern blotting analysis identified a 3.5-kb EcoRI fragment and a 14-kb HindIII fragment by hybridizing with the 32P-labeled probe, obtained by PCR labeling using the above primers for exon 1. The 14-kb fragment was purified and cloned. Restriction enzyme mapping and sequencing revealed that it contained 6.6 kb of the 5'-flanking region of the rat CRABP(II) gene. This 14-kb fragment was further digested with kpnI restriction enzyme to obtain a 4466-base fragment that contained the basic promoter region. This was ligated to the pGL3 basic luciferase reporter vector (Promega Co., Madison, WI). Further deletions of the –1354, –1211, –1182, –504 fragments were obtained by PCR amplification using the same reverse primer-added KpnI restriction enzyme site, (55/78) 5'-GGTACCGTACCTTGCTGTCCCCTT-3' and the forward primer-added KpnI restriction enzyme site, (–1354/–1337), 5'-GGTACCGCGATCCAGAAGCCCTTC-3' (–1210/–1193), 5'-GGTACCCAGTTCGACCCTCCACC-3' (–1177/–1160), 5'-GGTACCCACCAAAGCCTGTCAGTC-3' (–504/–487), 5'-GGTACCGAACAGAGCGACACCTCC-3', respectively. Each PCR product was digested by KpnI and cloned into the corresponding sites of the pGL3 basic vector. The right orientation was identified by PCR screening. Constructs 6 and 7 were generated by ligating the synthetic fragment of –1211/–1178 to the fragments of –799 or –754, respectively (Fig. 4). All the constructs created by PCR amplification were verified by sequencing.



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  		FIG. 4.
Identification of regions required for E2-stimulated transcription. Different constructs of the rat CRABP(II) upstream region (right panel) were cloned into the luciferase (Luc) reporter plasmid pGL3 basic vector as described under "Experimental Procedures." MCF-7 cells were cotransfected with the pGL3 gene constructs (1 µg) and ER expression plasmid (1 µg), together with pRL-TK vector as an inner control. After treatment of cells with ethanol or E2 (100 nM) for 48 h, luciferase activity was measured in cell lysates. Data are expressed as -fold induction by E2, relative to cells treated with ethanol alone, and are the average of three experiments (the range observed was less than 20%).

 

Cell Culture, Transient Transfection, and Luciferase Assay—MCF-7 cells obtained from the American Type Culture Collection (Manassas, VA) were routinely maintained in a humidified atmosphere containing 5% CO2. Cells were transiently transfected when they approached 70% confluence in 24-well plate using the SuperFect transfection reagent (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. The cells were cotransfected with 1 µg of estrogen receptor (ER) expression plasmid (ATCC) as the endogenous level of ER was low, and with 10 ng of pRL-TK vector to normalize the differences in transfection efficiencies. Following transfection, cells were changed to phenol red-free minimum Eagle's medium (Invitrogen) containing 10% dextran-coated charcoal-stripped fetal bovine serum, and cells were incubated with 100 nM E2 or vehicle for 2 days before harvesting for the luciferase assay. Cells were lysed using 1x passive lysis buffer, and 20 µl of lysate was assayed using Dual-Luciferase reporter assay system (Promega).

Electrophoretic Mobility Shift Assays (EMSA)—All the double strand oligonucleotides used for the EMSAs were prepared and high pressure liquid chromatography-purified by Qiagen Operon (Alameda, CA). The sequence of each sense oligonucleotide is shown in Table I. Oligonucleotides were labeled with [{gamma}-32P]ATP (10 Ci/ml) and T4 polynucleotide kinase. The EMSAs were performed as described previously (15). Briefly, 0.1–0.3 µg of the purified active form of ER protein (PanVera Co., Madison, WI) was first incubated in 20 µl of binding buffer containing 3 mM EDTA, 1 mM dithiothreitol, 2 µg of poly(dI-dC), and 4% Ficoll 400 at room temperature for 10 min, and then 1 ng of probe was added, and the sample was incubated another 15 min. For the competition experiments and supershift assay, 100–800-fold excess of competitor or 1–4 µg of ER antibody (PanVera Co., Madison, WI) was added to the reaction mixture, and the incubation was further continued at room temperature for 20 min. The reaction mixtures were submitted to electrophoresis on 4% polyacrylamide native gels. After running, the gel was dried and exposed to the Kodak film overnight.


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  		TABLE I
Sequence of the sense oligonucleotides used for the gel mobility shift assays

Underlining and boldface in the sequences indicates the half palindrome and changed bases, respectively.

 


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of CRABP(II) in Rat Uterus Was Induced Directly by E2 but Not by RA—Ovariectomized rats were administered either E2 or RA or vehicle alone and uteri were collected after 4 h. Uteri were also collected from ovariectomized rats that had received puromycin 30 min prior to administration of E2, RA, or vehicle. Analysis of the RNA obtained from these uteri by RPA revealed a potent induction (16–18-fold) of CRABP(II) message by E2 administration (Fig. 1). Puromycin had no effect on this increase, suggesting that the response to E2 did not require new protein synthesis and was sufficiently rapid to be a direct response.



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  		FIG. 1.
E2, but not RA, induced expression of the CRABP(II) gene in vivo. Total RNA (30 µg) from uteri of treated rats was analyzed by RPA for expression of CRABP(II) message (top), CRBP message (middle), and cyclophilin message (bottom). The -fold increase (X) from control (Con) is shown below each lane after normalization to measured cyclophilin message level. CRBP expression was monitored as a representative known RA-responsive gene. The data shown are representative of three similar experiments. P, puromycin.

 

Surprisingly, no increase in CRABP(II) message in the uterus was observed for animals administered RA, in contrast to observations for both the human and mouse CRABP(II) gene, albeit in different systems (11, 16). No increase was noted even at times as late as 48 h after RA administration (data not shown). To confirm that RA had indeed reached the uterus, the level of expression of cellular retinol-binding protein (CRBP), known to be directly regulated by RA (17), was determined and found to have increased about 5-fold in the animals receiving RA (Fig. 1). Thus, the lack of response of the rat CRABP(II) gene to RA could not be ascribed to delivery failure.

It was considered possible that an RA-induced expression might require exposure of the uterus to E2. To test this possibility, we injected ovariectomized rats with E2 and RA simultaneously and also provided RA 24 h after E2 treatment. When message levels were determined 4 h later, no effect of RA was observed over that of E2 alone (results not shown).

Analysis of the 5'-flanking Region of the Rat CRABP(II) Gene—Previous analyses of the 5'-flanking regions of the mouse and human CRABP(II) gene had not revealed any indication of an ERE. To see whether there might be a species difference in estrogen response, we isolated this region for the rat gene, as described under "Experimental Procedures." A 3.5-kb EcoRI fragment and a 14-kb HindIII fragment were identified (Fig. 2A). Restriction enzyme mapping analysis revealed that the 14-kb fragment contained about 6.6 kb of the 5'-flanking region (Fig. 2B). Digestion with KpnI produced a fragment that contained the TATA box and transcription start site but excluded the coding region.



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  		FIG. 2.
Isolation of the 5'-flanking region of the CRABP(II) gene. After a positive clone was digested with either EcoRI or HindIII, the digestion mixtures were separated on a 0.8% agarose gel. After transfer to membrane, a 3.5- and 14-kb fragment were identified from these two restriction enzymes, respectively, by using a partial sequence of exon I as probe (A). From further restriction enzyme mapping of the 14-kb fragment (shown in B), the fragment generated by kpnI was selected for further study.

 

The complete sequence of this 4.5-kb fragment was analyzed for known DNA binding protein recognition sites. The palindromic sequences of the canonical ERE (18) were not found. Putative regulatory elements for ubiquitous transcription factors such as AP1 (19), AP2 (20), and Sp1 (21) were evident in the proximal region of this fragment (Fig. 3). The proximal region shown corresponds to the published sequence of the mouse CRABP(II) 5' region, with which it has 85% sequence identity (13). Although we found no response of the CRABP(II) gene to RA in the uterus of the intact rat, there is a region corresponding to the mouse RA-response element mRARE2 but no correspondence to the reported mRARE1 (Fig. 3).



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  		FIG. 3.
Nucleotide sequence of the selected 4.5-kb fragment of the 5'-flanking region of the CRABP(II) gene. The nucleotides are numbered according to the transcription start site, which is indicated as +1. The TATA signal and ATG start codon are boxed; possible AP1, AP2, Sp1, and RARE binding sites and the sequence of the oligonucleotides used for P1 screening and the probe used for Southern blotting are underlined. Underlined EREc indicates the non-canonical ER binding site for CRABP(II) identified in this study. The imperfect consensus sequences are written in bold and italic. The position and sequence of the primers used for P1 screening and probe for Southern blot are shown in italic.

 

Promoter Activity of the 5'-flanking Region of the Rat CRABP(II) Gene—To determine whether the cloned 5'-flanking region of the rat CRABP(II) gene contained unidentified response elements allowing transcriptional activation by E2, various 5' end deletions were ligated into the pGL3-basic luciferase reporter vector (Fig. 4). The cell line chosen for testing these constructs was the MCF-7 human mammary carcinoma cell line as it expresses both CRABP(II) and the ER and, consequently, should contain any cell-specific factors involved in CRABP(II) expression. However, preliminary studies indicated that a better response was obtained if an ER expression vector was cotransfected and all experiments included that vector.

The longest fragment tested contained, with respect to the transcription start site, the region from –4411 to +77, ending 72 nucleotides prior to the initiation codon for translation (construct 1). After treatment with E2 for 48 h, the transcription activity of this fragment was about 13-fold higher than the basal promoter level (Fig. 4). Treatment with 1 µM RA gave no increase in transcription (data not shown). Various deletions from the 5' end of this fragment were examined, and induction remained at this level until –1210 (constructs 2 and 3). Deletion constructs shorter than –1210 were substantially lower, falling to a 2–3-fold increase over basal levels (constructs 4 and 5), indicating that the –1210 to –1178 region was critical for E2 response. The sequence of this region contained an imperfect consensus ERE, GCTCANNNCGACC, and an ERE half-site, TGTCA (Table I).

To examine whether the GC-rich regions, putative Sp1 binding sites, might also contribute to the ability of E2 to induce transcription, as has been observed for other genes, the –1210 to –1178 sequence was ligated to the proximal promoter region at position –799, just proximal to the second GC-rich sequence at –781 to –755 (construct 6). Induction was similar to that seen with the full-length construct (~8-fold versus ~12-fold.). However, ligation of the –1210 to –1178 fragment to the proximal region at position –754 reduced induction to the 2–3-fold increase seen when the –1210 to –1178 fragment was not present (construct 7; compare with construct 4). This suggested that the fragment –1210 to –1178 and the region –799 to –754 are both required for the E2-induced transcription of the rat CRABP(II) gene. The possible additional requirement of the more proximal GC-rich region was not examined.

We further examined the response of this promoter region to E2 in the rat hepatoma cell line H-4-II-E, which does not express CRABP(II) or the ER. The results were essentially the same for each construct as found in the MCF-7 cell line (data not shown), which suggested that no factor specific to a cell type was required in this in vitro deletion assay.

Test of the Putative ERE by ER Binding Studies—Known EREs are able to bind activated ER in vitro, as shown by electrophoretic mobility shift assays (EMSA). The –1210 to –1178 fragment, designated EREc (Table I), was tested for the ability to bind purified, activated ER. As a control, a sequence containing a canonical ERE was also used (wtERE).

When ER was incubated with 32P-labeled oligonucleotide EREc, a protein·DNA complex was formed, as revealed by EMSA (Fig. 5A, lane 1). This protein·DNA complex was confirmed to be an ER·DNA complex by the ability of ER antibody to supershift the complex in a dose-dependent manner (Fig. 5A, lanes 2–5).



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  		FIG. 5.
Binding of purified ER to CRABP(II) ERE (EREc) in gel mobility shift assays. A, antibody (Ab) supershift. 1 ng of 32P-labeled EREc was incubated with 0.3 µg of purified activated ER at room temperature for 15 min, the reaction mixture was run on 4% native polyacrylamide gel, and a protein·DNA complex was formed in lane 1. The ER·DNA complexes were shifted as indicated by the arrow when the different doses of ER antibody were applied to the reaction mixture for another 20 min at room temperature. B, competition of EREc binding to ER by unlabeled canonical ERE (wtERE). 1 ng of 32P-labeled EREc and 0.3 µg of purified activated ER were incubated at room temperature for 15 min, and then 100–400-fold molar excess of cold wtERE was added for another 20 min at room temperature, and the ER·EREc complex was competed away dose-dependently in lanes 2–5. There was no competitor added in lane 1. C, competition of wtERE binding to ER by unlabeled EREc or mutation of EREc (EREm). 1 ng of 32P-labeled wtERE and 0.1 µg of purified activated ER were incubated at room temperature for 15 min, and then 200–800-fold molar excess of cold EREc (lane 2–5) or 400 and 800-fold molar excess of cold EREm (lanes 6 and 7) was added. Lane 1 showed the ER·wtERE complex without adding any competitor.

 

As a further test of EREc as a potential ERE, gel mobility competition assays were performed with 100–400-fold molar excess of unlabeled wtERE over 32P-labeled EREc (Fig. 5B) and 200–800-fold molar excess of unlabeled EREc over 32P-labeled wtERE (Fig. 5C). A 400-fold excess of wtERE competed for all detectable binding of labeled EREc, whereas an 800-fold excess of EREc was required to compete for all detectable binding of wtERE. This suggested that the affinities of the ER for these sequences were similar but that the wtERE appears to bind more tightly to the ER. As a final test of specificity of binding of the ER to the EREc sequence, competition was tested for a 3-base mutant, EREm, with two base changes in the imperfect ERE region and one in the half-site (Table I). No apparent competition was observed for either a 400- or 800-fold excess. These results established that the –1120 to –1178 contained an imperfect ERE that can bind specifically to ER in vitro.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Previous work from our laboratory has shown that the expression of CRABP(II) in certain cells of the rat uterus and ovary correlates with the production of RA by those cells (2, 22). Recent studies by others have demonstrated an ability of the RA·CRABP(II) complex to translocate to the nucleus and mediate a direct transfer of RA to RARs, an ability not shared by the closely related protein CRABP (6, 7). The association of CRABP(II) with the nuclear receptor complex has been demonstrated to enhance the expression of RA-responsive genes (23).

In the work presented here, the demonstration that E2 directly induced the expression of CRABP(II) indicates that the effects of E2 on a particular tissue/cell may well result in the induction or modulation of expression of RA-responsive genes, in addition to E2-responsive genes. This would appear to greatly increase the number of genes accessible by the E2 signal and provides a direct link between the action of a steroid hormone and the action of RA.

Interestingly, we saw no change in expression of the endogenous CRABP(II) gene or of the reporter constructs when RA was provided. Previously, we had observed that E2 administration to the prepubertal female rat led to acquisition of the ability of the lining epithelium to synthesize RA as well as to express CRABP(II). That expression of CRABP(II) would then appear to be independent of RA synthesis by those cells. This result is different from other studies in which CRABP(II) gene expression was strongly increased by RA either in human skin in vivo or in cultured human skin fibroblasts in vitro (24). RA also was shown to increase expression of CRABP(II) in the F9 murine cell line (16). These differences might be explained by species or cell-type differences. However, it was observed that this increase of expression in both intact human skin and in the F9 cell line was blocked by inhibition of protein synthesis, suggesting that the effect was indirect. Nuclear run-on experiments suggested that the increase was controlled by a post-transcriptional mechanism (25). Still, we observed no increase in CRABP(II) expression at times up to 48 h, suggesting that even an indirect regulation by RA was not occurring in the rat uterus under the conditions examined here.

Analysis of the 5'-flanking region of the rat CRABP(II) gene showed 85% sequence identity to the proximal 1 kb of the corresponding sequence reported for the mouse gene, including an identical DR1 mRARE2 repeated motif (13) and some ubiquitous transcription factor binding sites, such as Sp1, AP1, and AP2 (fig. 3), but there was no sequence corresponding to an unidentified functional DR2 mRARE1. It should be noted that this region of the mouse promoter did lead to RA-stimulated transcription of a reporter gene in transfected cells, whereas we did not observe such an increase. This may explain why RA has no effect on rat CRABP(II) gene expression in vivo or when transfected in MCF-7 cells in vitro and may suggest a species difference in regulation by RA. However, it should be stressed that the actual direct regulation of the native gene in both human and murine cells by RA remains in question.

No obvious ERE was noted in the analysis of the 4.5-kb fragment, but it is well established that E2-directed gene regulation can be quite complex. Many naturally occurring copies of the 13-base pair consensus ERE are imperfect (26–31). Also, several genes activated by E2 involve both ERE and GC-rich Sp1 binding sites, such as creatine kinase B (32), bcl-2 (33), uteroglobin (34), insulin-like growth factor-binding protein-4 (35), transforming growth factor {alpha} (36), RAR{alpha} (37), and LDL receptor (38), or ERE interaction with USF-1 and USF-2 as in the cathepsin D promoter (39). That proved to be the case here as well. The identified region that bound the ER contained three imperfect half-palindromic EREs, not obvious from sequence analysis alone. In addition, a possible Sp1 binding site was required to obtain the E2 response in vitro. Thus, this appears to be another version of a complex E2-responsive promoter.

Our preliminary studies2 indicate that E2 also directly regulates expression of the enzymology of RA synthesis. It will be of interest to examine whether this regulation shares features observed for the CRABP(II) gene.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY226560 [GenBank] .

* This work was supported by National Institutes of Health Grants HD25206 and DK32642. Core facilities used were from the Diabetes Center (oligonucleotide synthesis) and the Vanderbilt-Ingram Cancer Center (DNA sequencing), supported by National Institutes of Health Grants DK 20593 and CA 68485, respectively. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University, 23rd Ave. at Pierce, Nashville, TN 37232. Tel.: 615-322-6331; Fax: 615-343-7347; E-mail: david.e.ong{at}vanderbilt.edu.

1 The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RARE, RA-response element; ER, estrogen receptor; ERE, estrogen-response element; CRABP(II), cellular retinoic acid-binding protein(II); E2, 17{beta}-estradiol; CRBP, cellular retinol-binding protein; RPA, RNase protection assay; EMSA, electrophoretic mobility shift assay; wt, wild type. Back

2 X.-H. Li and D. E. Ong, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr Hyekyung Cho for significant technical help and discussions on this study, Dr Michael H. Melner and Dr Susan J. Ruff for discussions and comments, and Dr Xiaohui Wang for kindly providing us RAR{alpha} expression vector and H-4-II-E cells.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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