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J. Biol. Chem., Vol. 278, Issue 37, 35819-35825, September 12, 2003
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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.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-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 |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, , and 
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.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 [-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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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 [-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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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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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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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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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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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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
(36), RAR
(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.
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FOOTNOTES |
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* 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. 
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-estradiol; CRBP, cellular retinol-binding protein; RPA, RNase
protection assay; EMSA, electrophoretic mobility shift assay; wt, wild
type.
2 X.-H. Li and D. E. Ong, unpublished results.
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ACKNOWLEDGMENTS |
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expression
vector and H-4-II-E cells. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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