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Molecular Endocrinology 13 (8): 1225-1236
Copyright © 1999 by The Endocrine Society

The Promoter of the Rat 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene Contains a Tissue-Specific Estrogen-Responsive Region

Luciano Di Croce, Guillermo P. Vicent, Adali Pecci, Giovannella Bruscalupi, Anna Trentalance and Miguel Beato

Institute for Molecular Biology and Tumor Research (IMT) D-35037 Marburg, Germany
Department of Cellular and Developmental Biology Università "La Sapienza" 00185 Rome, Italy
Department of Biology Università di Roma 3 00146 Rome, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The isoprenoid metabolic pathway is mainly regulated at the level of conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonate, catalyzed by HMG CoA reductase. As estrogens are known to influence cholesterol metabolism, we have explored the potential regulation of the HMG CoA reductase gene promoter by estrogens. The promoter contains an estrogen-responsive element-like sequence at position -93 (termed Red-ERE), which differs from the ERE consensus by one mismatch in each half of the palindrome. A Red-ERE oligonucleotide specifically bound estrogen receptor in vitro and conferred receptor-dependent estrogen responsiveness to a heterologous promoter in all cell lines tested. However, expression of a reporter driven by the rat HMG CoA reductase promoter was induced by estrogen treatment after transient transfection into the breast cancer cell line MCF-7 cells but not in hepatic cell lines expressing estrogen receptor. Estrogen induction in MCF-7 cells was dependent on the Red-ERE and was strongly inhibited by the antiestrogen ICI 164,384. A functional cAMP-responsive element is located immediately upstream of the Red-ERE, but cAMP and estrogens inhibit each other in terms of transactivation of the promoter. Similarly, induction by estrogens was inhibited by micromolar concentrations of cholesterol, likely acting via changes in occupancy of the sterol-responsive element located 70 bp upstream of the Red-ERE. Thus, within its natural context, Red-ERE is able to mediate hormonal regulation of the HMG CoA reductase gene in tissues that respond to estrogens with enhanced cell proliferation, while it is not operative in liver cells. We postulate that this tissue-specific regulation of HMG CoA reductase by estrogens could partially explain the protective effect of estrogens against heart disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isoprenoid biosynthesis provides the cell with essential compounds such as cholesterol, dolichols, ubiquinon, and prenylated proteins. The common precursor of these compounds, mevalonate, is produced by the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (1), a 97-kDa glycoprotein that resides in the endoplasmatic reticulum, where the enzyme is also degraded (2). Cholesterol represents the predominant product of the biosynthetic pathway and plays a primary role in membrane biogenesis and in steroid hormone biosynthesis. The other isoprenoid compounds produced in branched paths of the main biosynthetic pathway are involved in protein glycosylation (dolichol), energetic processes (ubiquinon), cell membrane traffic (Rab), and nuclear structure (lamins) (3). The level of HMG CoA reductase can be modulated at the level of gene transcription (4, 5), mRNA stability (6, 7), translation (8, 9), and/or enzyme degradation (3, 10). Furthermore, the specific activity of HMG CoA reductase is reversibly regulated by phosphorylation/dephosphorylation mechanisms and by oxidation events (11).

To maintain an adequate cholesterol concentration, the cell changes the level of the endogenous synthesis and exogenous supply through a feedback regulation of HMG CoA synthase, HMG CoA reductase, and low-density lipoprotein receptor (LDLr). Regulation of the HMG CoA reductase controls the endogenous cholesterol production, while LDLr controls the exogenous uptake as well as the supply to the extrahepatic tissues (3). The sterol-regulatory element (SRE) present in the promoters of these genes mediates the transcriptional regulation by sterols (12, 13). The transcription factors that bind this element at low cholesterol concentrations (SRE-binding proteins, SREBPs) have been characterized (14, 15, 16). However, metabolic changes detectable in different physiological states suggest the occurrence of additional regulatory mechanisms involving several hormones. For instance, TSH is reported to induce the transcription of the HMG CoA reductase gene via cAMP (17).

Estrogens have been strongly connected to the very low incidence of heart disease in women and with the hypolipidemic effect caused by an enhanced LDLr function. Furthermore, estrogens have been reported to affect the metabolism of isoprenoid compounds in various species displaying species-specific differences (18, 19, 20, 21). The variable effects on the HMG CoA reductase observed in the rat liver after estrogen treatment could be explained by differences in the circadian rhythm and feeding status of the experimental animals (22, 23, 24). In our hands, estrogen administration to rats caused an early increase of LDLr and a late increase of HMG CoA reductase, detectable only after 5 days (24). In vitro experiments showed that estrogens induce HMG CoA reductase activity as well as cholesterol synthesis (25, 26). Direct transcriptional and posttranscriptional mechanisms have been proposed to explain the estrogen effects (27), even though indirect mechanisms involving changes in cholesterol content acting via SRE or by changes in second messenger levels (cAMP, Ca++, or inositol triphosphate) cannot be excluded (28).

Little is known about the molecular mechanism(s) of estrogen effects on isoprenoid metabolism. It is generally accepted that estrogens act by interacting with their intracellular receptor, the estrogen receptor (ER), which is a ligand-dependent member of the nuclear receptor family (29, 30). The ER binds to specific DNA sequences, called estrogen-responsive elements (EREs), that share the palindromic consensus sequence GGTCAnnnTGACC (31) and are located in the promoter or enhancer regions of many estrogen-regulated genes. To explore the possibility of a direct effect of estrogenic hormones on the HMG CoA reductase gene, we screened the sequence of the rat gene for the presence of potential EREs. Starting at position -93, we found an ERE-like sequence, CGTCAGGCTGAGC (hereafter denoted Red-ERE for HMG CoA reductase-ERE). Here we show that ER binds specifically to the Red-ERE and that the isolated Red-ERE confers estrogen responsiveness to a heterologous promoter in all cell lines tested. Thus, the Red-ERE behaves as a bona fide ERE. However, a reporter plasmid carrying the native HMG CoA reductase promoter sequences up to -323, including the Red-ERE, is transactivated by estrogens in the breast cancer cell line MCF-7, but not in two hepatic cell lines transfected with ER expression vectors. The activity of the Red-ERE is modulated by adjacent cis-acting elements, in particular by a sterol-responsive element and a cAMP-responsive element. Our results are compatible with a function of the Red-ERE in mediating hormonal induction of the rat HMG CoA reductase gene in peripheral tissues that respond to estrogens with cell proliferation. Estrogen regulation is tissue-specific as it does not operate in hepatic cell lines expressing a functional ER.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Red-ERE Binds ER{alpha} in Vitro
A computer comparison of the promoter sequences of human, hamster, mouse, and rat HMG CoA reductase genes showed a high degree of homology (Fig. 1Go), in particular in the region of the SRE, and revealed the presence of a potential estrogen responsive element (ERE) close to a cAMP-responsive element (CRE) (Fig. 1Go). The ERE-like sequence in the rat HMG CoA reductase gene promoter (Red-ERE) between -81 and -93 (CGTCAGGCTGAGC, see Fig. 1Go) is well conserved and presents two mismatches (bases underlined) when compared with the palindromic consensus ERE identified within the Xenopus laevis vitellogenin A2 gene (cons-ERE; GGTCAnnnTGACC) (31).



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  		Figure 1. Alignment of HMG CoA Reductase Promoter Sequences from Human, Hamster, Mouse, and Rat

The rat HMG CoA reductase promoter from -277 to +63 is shown aligned with the corresponding regions of the human, hamster, and mouse promoters. The SRE, the CRE, and the potential ERE are boxed. The asterisks denote conserved bases, while the diamond denotes the major transcription start site.

 
To test whether the putative Red-ERE is able to bind ER in vitro, we performed band shift experiments using nuclear extracts from the estrogen-responsive MCF-7 cell line and an oligonucleotide including HMG CoA promoter sequences from -98 to -77 (Red-ERE). The consensus ERE from the Xenopus vitellogenin A2 gene (32, 33) was used as positive control. A complex with the same retarded mobility as with the consensus ERE (cons-ERE) was detected with the Red-ERE oligonucleotide (Fig. 2AGo, lanes 1 and 3). Both complexes were competed by an excess of the specific, but not unspecific, unlabeled oligonucleotides (Fig. 2AGo, lanes 2 and 4, and data not shown). Densitometric analysis showed that ER binds with higher affinity to the cons-ERE than to the Red-ERE (see below).



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  		Figure 2. Specific Binding of ER to the Red-ERE and Its Relative Affinity

A, Nuclear extracts from MCF-7 cells were incubated with a 32P-radiolabeled oligonucleotide containing the cons-ERE (lanes 1 and 2) or the Red-ERE (lanes 3 and 4), and specific binding was determined by EMSA. Competition with a 200-fold molar excess of cons-ERE (lane 2) or Red-ERE (lane 4) was performed as specificity controls. The arrow indicates the ER/DNA complexes. B, EMSA was performed using either recombinant ER (lanes 1–7) or MCF-7 nuclear extract (MCF-7 NE; lanes 9 and 10). Equal amounts of 32P-labeled Red-ERE (lanes 1, 4, and 5), cons-ERE (lanes 2 and 6–10) or PRE (lane 3) were added to each reaction with a 200-fold molar excess of competitor Red-ERE (lanes 4 and 6) or competitor cons-ERE (lanes 5, 7, and 10). The arrow indicates the ER/DNA complexes. C, The ability of the Red-ERE to compete with the 32P-labeled cons-ERE for binding to the ER was analyzed by adding increasing amounts (0, 10, 20, 100, 200, and 500-fold molar excess) of competitor Red-ERE. The arrow indicates the ER/cons-ERE complexes. D, 32P-labeled cons-ERE was incubated with increasing amounts (0, 0.2, 0.4, 2, 4, and 10 pmol) of either cons-ERE or Red-ERE to measure their relative affinities to ER in nuclear extracts of MCF-7 cells. The amount of the complexes ER/cons-ERE in the absence of competitor was set at 100%. The SE was less than 3%.

 
The retarded complexes observed with nuclear extracts were likely due to binding of ER as a similar complex could be formed using recombinant ER{alpha} expressed in baculovirus-infected insect cells (34) (Fig. 2BGo, compare lanes 1 and 2 with 9), and a supershift was observed in the presence of an antibody specific for ER{alpha} (data not shown). Moreover, formation of these complexes was also effectively inhibited by addition of an excess of unlabeled cons-ERE or Red-ERE oligonucleotides (Fig. 2BGo, lanes 4–7). Importantly, the recombinant ER{alpha} did not bind an oligonucleotide containing the related sequence of a progesterone-responsive element (PRE) (Fig. 2BGo, lane 3), demonstrating its specific binding to the ERE oligonucleotides. These experiments confirm the involvement of ER in the retarded complexes and indicate the presence of an heretofore undetected ER-binding site in the promoter of the rat HMG CoA reductase gene.

Relative Affinity of ER{alpha} for Red-ERE and cons-ERE
Since the Red-ERE exhibits two mismatches when compared with the cons-ERE, it was important to determine whether the Red-ERE was able to compete for the binding of the ER to the cons-ERE. We performed binding experiments with MCF-7 nuclear extracts using the labeled cons-ERE and increasing amounts (10- to 500-fold molar excess) of unlabeled Red-ERE (Fig. 2CGo). The specifically retarded band was significantly reduced in the presence of a 20-fold excess (Fig. 2CGo, lane 3) and was almost abolished by a 500-fold excess of unlabeled Red-ERE (Fig. 2CGo, lane 6).

To determine the relative affinities, we compared the ability of unlabeled cons-ERE and Red-ERE oligonucleotides to compete for the binding of ER to the labeled cons-ERE sequence (Fig. 2DGo). The cons-ERE oligonucleotide competed very effectively for ER binding: a 50% reduction in ER-DNA complex formation was observed with 0.4 pmol of the unlabeled cons-ERE oligonucleotide. In contrast, about 0.9 pmol of the Red-ERE oligonucleotide was required to reduce by 50% binding of ER to the labeled cons-ERE. These results indicate that ER binds to the Red-ERE with approximately half the affinity as to the consensus ERE.

Red-ERE Confers Estrogen Responsiveness to a tk-Minimal Promoter in All Tested Cell Lines
We next investigated whether, as reported for a cons-ERE, the Red-ERE is able to confer estrogen responsiveness to a heterologous promoter. To this aim, a single copy of either the cons-ERE or the Red-ERE was inserted in front of a minimal herpes simplex virus thymidine kinase (tk) promoter linked to the firefly luciferase gene and transiently transfected in Cos-1 cells along with an expression vector for ER{alpha} (Fig. 3AGo). The cells were incubated with 100 nM of either 17ß-estradiol or the pure steroidal antiestrogen ICI 164,384. The level of promoter activity in cells treated with the solvent alone (ethanol, EtOH) was 10–15% higher than the value observed in cells treated with ICI. As a negative control, we used the wild-type tk promoter, which did not respond to hormone treatment. In contrast, estradiol induced the expression of the transfected construct containing the cons-ERE (pTK-cons-ERE) by about 12-fold, whereas the Red-ERE containing reporter (pTK-Red-ERE) was induced about 5-fold. This difference in induction efficiency correlates with the observed lower affinity of ER for the Red-ERE compared with the cons-ERE (see above).



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  		Figure 3. Influence of Estrogen on HMG CoA Reductase Promoter Activity in Mammary and Liver Cells

A, Vectors containing a single copy of Red-ERE or cons-ERE in front of a tk minimal promoter driving the luciferase gene (pTK-cons-ERE or pTK-Red-ERE, respectively) were transfected into either Cos-1 cells or HepG2 cells, along with expression vector for the human ER (pHEGO). The cells were incubated for 48 h with 100 nM ICI 164,384 (ICI) or 100 nM 17ß-estradiol (Estrogen) or with solvent alone (ethanol, EtOH). The luciferase activity (expressed in arbitrary units) is shown relative to the activity in cells treated with EtOH; the values represent the mean of four independent determinations calculated after normalization for cotransfected pRSV-lacZ. The SE was less than 3%. The empty expression was used as a negative control (pTK). B, CAT assays were performed using MCF-7 cells transfected with the plasmid pHMGRed-CAT containing sequences of the HMG CoA reductase promoter (-323 to +442) linked to a CAT reporter gene. Cells were treated with solvent alone (ethanol, EtOH) or with 100 nM ICI 164,384 or with 17ß-estradiol (indicated by E) at concentrations of 100 nM or 10 nM, as indicated. CAT activity is shown relative to the activity in cells treated with ICI. The values represent the mean ± SE of three independent determinations calculated after normalization for the cotransfected reference plasmid pRSV-luc. C, Plasmid containing either the wild-type construct (pHMGRed-luc) or the Red-ERE-deleted promoter (pHMGRed{Delta}ERE) was transfected into MCF-7 cells or HepG2 cells. The cells were incubated with solvent alone (ethanol, EtOH) or with 100 nM ICI 164,384 (ICI) or with 100 nM 17ß-estradiol (Estrogen). The values shown represent the mean ± SE of three separate determinations calculated after normalization for the cotransfected reference plasmid pRSV-lacZ.

 
The Red-ERE-tk reporter was also tested in liver cells (HepG2) cotransfected with an expression vector for ER{alpha}. As in Cos-1 cells, we reproducibly observed a 4-fold response to estrogens that was lower than the response found with the cons-ERE-tk reporter (Fig. 3AGo). The Red-ERE-tk reporter was also induced by estrogens when transfected in MCF-7 cells (data not shown). Our results confirm the ability of the isolated Red-ERE to confer estrogen responsiveness to a heterologous promoter in various cell types and, therefore, identify the corresponding sequence as a bona fide ERE.

The rat HMG CoA Reductase Gene Promoter Is Induced by Estrogens in MCF-7 Cells but Not in Hepatic Cell Lines
To investigate whether the Red-ERE is functional within the context of the HMG CoA reductase gene promoter, we used the reporter plasmid pHMGRed-CAT containing the chloramphenicol acetyl transferase (CAT) gene of Escherichia coli driven by a rat promoter fragment spanning from 323 bp upstream to 442 bp downstream of the major transcription start site. This reporter was transiently transfected in the estrogen-responsive MCF-7 cell line. Figure 3BGo shows that the HMG CoA reductase gene promoter responded to 17ß-estradiol stimulation in a dose-dependent manner. The average induction was 2-fold at 10 nM and 3-fold at 100 nM of 17ß-estradiol when compared with cells treated with the antiestrogen ICI. A similar estrogen response was observed in a rat endometrial cell line that express ER (35) (data not shown). Deletion of the potential ERE element (pHMGRed{Delta}ERE-luc) completely eliminated the hormone-dependent transactivation of the promoter but had no influence on the activity of the reporter in the presence of ethanol or antiestrogen (Fig. 3CGo). These results demonstrate the estrogen responsiveness of the rat HMG CoA reductase gene promoter and are compatible with the notion that the hormonal effect is mediated by the Red-ERE sequence.

Since the liver is the main tissue involved in the body cholesterol homeostasis, we next investigated the effects of estrogen administration on the HMG CoA reductase promoter transiently transfected in two different hepatic cell lines: the human HepG2 cell line and the rat Fto cell line. As these cells do not contain sufficient levels of endogenous ER, an expression vector for ER{alpha} was cotransfected. In neither case did we observe an effect of estrogens on expression of the HMG CoA reductase promoter (Fig. 3CGo and data not shown) although the transfected ER{alpha} was shown to be functionally active on either a canonical ERE reporter or on the Red-ERE in front of a heterologous promoter (Fig. 3AGo) and activated the HMG CoA reductase gene promoter in Cos-1 cells (Fig. 3CGo). Note that the basal activity of the HMG CoA reductase promoter in hepatic cells was consistently 8- to 10-fold higher than in other cell lines, as previously observed (36). This could be explained by the presence in different cells of different ratios of SREBP isoforms (14, 37) or by the presence of specific hepatic transcription factor(s). Thus, the HMG CoA reductase promoter is selectively responsive to estrogens in estrogen target cells, such as mammary and endometrial cell lines, but not in hepatic cell lines.

Modulation of ERE Function by Cholesterol Levels
The HMG CoA reductase promoter encompasses an SRE that is known to mediate regulation of the promoter in response to changes in cholesterol levels (12) and to be the target of SREBP (38). To explore a possible cross-talk between these signaling pathways and estrogen regulation, we tested the effect of cholesterol on hormone-independent and estrogen-induced expression of the promoter in transfection assays. In MCF-7 cells, addition of 25 µM 25-OH-cholesterol to the medium had a weak inhibitory effect on promoter activity in the presence of antiestrogen but caused an almost complete inhibition of estrogen transactivation (Fig. 4AGo). In hepatic cells the same concentration of cholesterol caused a 50% reduction in HMG CoA reductase promoter activity, and the promoter remained unresponsive to estrogen-activated ER{alpha} (data not shown). Interestingly, when a down-mutation of the SRE site was introduced in the HMG CoA promoter ({Delta}SRE), cholesterol addition failed to prevent estrogen transactivation in MCF-7 cells (Fig. 4AGo). These results confirm the strong inhibitory influence of signals acting via the SRE on transcriptional regulation of the rat HMG CoA reductase promoter and furthermore demonstrated that these signals can modulate the inductive action of estrogens (3, 38).



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  		Figure 4. Influence of Cholesterol and cAMP on the Estrogen Induction of HMG CoA Reductase Promoter

A, MCF-7 cells were transfected with a reporter plasmid containing the CAT gene under the control of the rat HMG CoA reductase promoter, either wild-type (pHMGRed-luc) or carrying the SRE deletion ({Delta}SRE). Cells were treated with solvent alone (ethanol, EtOH) or 100 mM of ICI 164,384 (ICI) or 17ß-estradiol (E), in the presence or absence of 25 µM 25-OH-cholesterol as indicated below each bar. Values represent CAT activity in arbitrary units. The mean ± SE values from three independent determinations were calculated after normalization for cotransfected pRSV-lacZ. B, MCF-7 cells were transfected with a reporter plasmid containing the CAT gene under the control of the rat HMG CoA reductase promoter and treated with solvent alone (ethanol, EtOH), 100 nM ICI 164,384 (ICI), 100 nM 17ß-estradiol (E), 1 mM (Bu)2cAMP (cAMP), or various combinations as indicated below each bar. Values represent CAT activity expressed in arbitrary units and represent the mean ± SE calculated from four separate determinations after normalization for cotransfected pRSV-lacZ. C, MCF-7 cells were cotransfected with a plasmid containing the wild-type HMG CoA reductase promoter, and with a plasmid carrying four tandem copies of a canonical CRE (p4xCRE), and treated with 100 nM 17ß-estradiol (E) as indicated below each bar. The values represent CAT activity expressed in arbitrary units and represent the mean ± SE from four separate determinations calculated after normalization for cotransfected pRSV-lacZ. D, EMSA with 32P-labeled oligonucleotide encompassing both the Red-ERE and CRE sequences and nuclear extracts from MCF-7 cells. A 200-fold excess of Red-ERE (lane 2), cons-ERE (lane 3), or CRE (lane 4) was added as competitor. The top arrow indicates the ER/DNA complex while the lower arrow indicates the CRE-containing retarded complex.

 
Modulation of ERE Function by cAMP
The sequence TGACGTAG (17), which is very similar (with only two mismatches) to the consensus CRE (39), is found immediately upstream of the Red-ERE in the HMG CoA reductase promoter. Since cAMP is known to influence the expression of the HMG CoA reductase gene (17), we tested its influence on the activity of the pHMGRed-CAT reporter in the presence or absence of estrogens and antiestrogens (Fig. 4BGo). (Bu)2cAMP (1 mM) alone induced the activity of the reporter to about the same level as 100 nM 17ß-estradiol. However, simultaneous addition of cAMP and 17ß-estradiol did not lead to a further increase in activity, suggesting that the two inducers cannot act additively or synergistically. In fact, the two inducers appear to compete, as shown by the results obtained in the presence of the pure antiestrogen ICI, which leads to a further increase in cAMP induction of the reporter (Fig. 4BGo). This suggests that the effect of cAMP may be inhibited in part by remaining estrogenic activities in the culture medium, or by a ligand-independent activation of ER.

To test whether estrogen induction is inhibited by the basic activity of the cAMP-signaling pathway, we performed oligonucleotide competition studies. MCF-7 cells were cotransfected with the pHMGRed-CAT reporter plasmid along with an excess of a plasmid carrying four tandem copies of a canonical CRE (p4xCRE), which should bind endogenous CRE-binding protein (CREB) and thus prevent its interaction with the CRE on the transfected HMG CoA promoter (Fig. 4CGo). Whereas this plasmid had little effect on the activity of the HMG CoA reductase reporter in the absence of estrogens, it doubled the estrogen response, increasing it from 3-fold in the absence of the CRE plasmid to 6-fold in its presence (Fig. 4CGo). These findings are compatible with an inhibitory effect of endogenous cAMP levels on estrogen induction of the HMG CoA reductase promoter.

To explore the mechanism of the mutual inhibition, we performed electrophoretic mobility shift assays (EMSAs) with a DNA fragment encompassing both the Red-ERE and CRE sequences. In the absence of competitor DNA, two complexes with different mobilities were observed using MCF-7 nuclear extract (Fig. 4DGo, lane 1). The slower complex likely corresponded to ER binding, as it was effectively reduced by the addition of unlabeled Red-ERE or cons-ERE (Fig. 4DGo, lanes 2 and 3, respectively). The faster complex probably involved proteins interacting with the CRE, since it was markedly reduced by an excess of unlabeled CRE (Fig. 4DGo, lane 4). Under no conditions did we find indications for a ternary complex containing both ER- and CRE-associated factors. Thus, both sites are occupied independently and no indication for simultaneous or cooperative binding is found. Our data are compatible with a mutually exclusive binding of ER and CREB, or related factors, to the HMG CoA reductase promoter region.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The nucleotide sequence of rat HMG CoA reductase promoter is highly conserved between species (see Fig. 1Go). This conservation is 84% over the regulatory region that includes SRE, CRE, and ERE, suggesting an important role for all of these sites in regulating expression of the HMG CoA reductase gene. The involvement of the CRE site (17) as well as the SRE site (38) in regulation of the gene is well documented. Here, we have investigated the molecular basis for estrogen regulation of HMG CoA reductase gene expression. The Red-ERE is able to bind specifically ER from MCF-7 nuclear extracts as well as recombinant ER{alpha}. The observation that the retarded complexes exhibit the same mobility on the Red-ERE as on the cons-ERE and that it is specifically supershifted in the presence of monoclonal antibody confirm that a homodimer of ER{alpha} binds to the HMG CoA reductase promoter. Quantitative competition experiments reveal that ER{alpha} binds the Red-ERE with 50% of the affinity observed for the consensus ERE, likely reflecting the two mismatches present in the Red-ERE.

The Red-ERE not only binds ER but also acts as a functional ERE in front of a heterologous promoter when transfected in a variety of cells, including two hepatic cell lines. The Red-ERE is functional in the context of the HMG CoA reductase promoter in mammary and endometrial cells. Estrogens (10–100 nM) induce the expression of the promoter in MCF-7 cells, and this effect is abolished by mutations of the Red-ERE. However, whereas the estrogen induction of the isolated Red-ERE was 6-fold, the response of the native HMG CoA reductase promoter was only 3-fold, suggesting that other sequences within the promoter interfere with ERE function.

Since the original discovery of an ERE in the Xenopus vitellogenin A2 gene (31), only one other gene has been found containing a 100% identical ERE element (40). In contrast, several estrogen-responsive genes have been shown to utilize degenerate EREs (41, 42, 43, 44, 45). Thus, it seems that imperfect EREs are more frequently used than consensus ERE, although canonical EREs confer stronger activation by estrogens. One possible reason for the higher frequency of imperfect EREs is that they may facilitate fine tuning of the hormone induction by other signal transduction pathways impinging on nearby regulatory elements of the promoter.

In contrast with the results obtained with classical estrogen target cells, like mammary and endometrial cells, the HMG CoA reductase promoter does not respond to estrogen treatment in two hepatic cell lines. This is not due to a defect of the transfected ER, since in these cells, the isolated Red-ERE is able to confer estrogen responsiveness to the tk promoter. Thus, the Red-ERE is potentially functional in liver cells but is inactive in the context of the HMG CoA reductase promoter. These data suggest a molecular explanation for the lack of estrogen induction of cholesterol biosynthesis in liver cells in contrast to MCF-7 cells (46).

Among other potential regulatory elements, the HMG CoA reductase promoter contains a SRE and a CRE element. The SRE seems to be functional in mammary cell lines since addition of cholesterol inhibited the estrogen response of the promoter, and this effect was dependent on the integrity of the SRE site. Thus, feedback inhibition by cholesterol appears to play a dominant role in the regulation of the HMG CoA reductase promoter. Transient transfection experiments demonstrated that estrogens and the signals acting via the CRE do not synergize but, on the contrary, appear to inhibit each other. Using a fragment of the HMG CoA reductase promoter encompassing the ERE and CRE sequences, no simultaneous binding of CREB and ER{alpha} was detected. As the two elements are separated by only 2 bp, steric hindrance could preclude simultaneous binding of ER and CREB to the promoter (47). A similar scenario has been postulated to explain the lack of simultaneous binding of progesterone receptor and NF1 to the mouse mammary tumor virus (MMTV) promoter DNA (48). Alternatively, the cross-talk can be mediated by complex interaction with the integrator CBP/p300, which is known to interact directly with ER (49), CREB (50), and SREBP (51) and exhibits histone acetyltransferase activity (52).

Our results with liver cell lines are in good agreement with our previous observations that while estrogen treatment in vivo affects the protein level of HMG CoA reductase in rat liver, it does not modify the level of mRNA (24). The lack of induction either in vivo (rat liver) or in vitro (hepatic cell lines) could result from interplay between the estrogen and other signal transduction pathways targeting the HMG CoA reductase promoter (53). Here both the SRE and/or the CRE could play a role. The inhibition by cholesterol of the estrogen induction of the HMG CoA reductase promoter observed in transfection assays offers a possible explanation for the lack of hormonal induction in hepatocytes. The increased intracellular cholesterol level observed in liver after estrogen treatment (24, 54), and the estrogen induction of the LDL receptor expression, could exert a feedback-inhibitory effect on transcription of the HMG CoA reductase gene by modifying the ratio of different SREBP isoforms present in liver cells (14, 37). On the other hand, Osborne and co-workers (4) have shown that the CRE site in the HMG CoA reductase promoter is strongly protected from deoxyribonuclease I (DNaseI) digestion by nuclear liver proteins. Deletion of this region almost completely abolished transcription. In contrast, no major effect was observed when the region covering the ERE site was deleted. These results are in agreement with our finding that the ERE is not active in liver and suggest that cAMP may be a main regulator in this tissue. Alternatively, in hepatocytes, the high basal expression level of the HMG CoA reductase promoter (10-fold) might preclude any further physiological induction.

Heart disease has been related to an increase in blood cholesterol levels and subsequent deposit in the vascular walls. If estrogens were able to induce the HMG CoA reductase promoter in the liver, the cholesterol flux from the liver would increase in response to estrogens. However, we demonstrated here that HMG CoA reductase is not estrogen inducible in liver cells, thus allowing estrogens to exert other protective effects in peripheral tissues (e.g. vasodilatation, heart activity moderation) without affecting cholesterol production. Additionally, in estrogen target cells, such as mammary cells and endometrial cells, the induction of the HMG CoA reductase expression would facilitate cholesterol synthesis that may be required for the proliferative response of these cells to estrogens. Thus, the difference in estrogen responsiveness of the HMG CoA reductase gene in liver and classical estrogen target cells could explain in part of the low incidence of coronary heart disease in females (55). However, as the ERE in the human HMG CoA reductase promoter exhibits an additional mismatch, experiments with the human promoter as well as studies on the estrogen regulation of the endogenous HMG CoA reductase gene in mammary gland would be required to support the physiological significance of our findings.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals and Materials
Cell culture media and sera were purchased from Life Technologies, Inc. (Gaithersburg, MD). [14C]-Chloramphenicol and [32P]-{gamma}-ATP were from Amersham Pharmacia Biotech (Arlington Heights, IL). 17ß-Estradiol, 25-OH-cholesterol, and (Bu)2cAMP were purchased from Sigma Chemical Co. (St. Louis, MO). ICI 164,384 was kindly provided by A. E. Wakeling, Zeneca Pharmaceuticals. Restriction enzymes were purchased from Roche Molecular Biochemicals (Foster City, CA).

Reporter Plasmids
The plasmid pHMGRed-CAT, containing a 765 bp PstI fragment (from -323 to +442) of the rat HMG CoA reductase promoter cloned into the pEMBL-8-CAT vector, was kindly provided by M. Bifulco. For construction of the luciferase reporter, pHMGRed-luc, the PstI fragment of pHMGRed-CAT, was cloned into the SmaI site of the pXP2 vector (56). p{Delta}HMGRed-luc was generated by deleting the Red-ERE. Briefly, pHMGRed-luc was linearized with EspI, which cleaves at the center of the Red-ERE, and subsequently treated with S1 nuclease (1 U/µg DNA for 30 min at 37 C) before religation. The resulting plasmid, p{Delta}HMGRed-luc, lacked the ERE as confirmed by DNA sequence analysis. pERETK-luc and pRedTK-luc were generated by cloning oligonucleotides containing the ERE-consensus (5'-GATCCGTCAGGTCACAGTGACCTGATG-3') or the Red-ERE (5'-TAGGCCGTCAGGCTGAGCAGCC-3'), respectively, into the SmaI site of the pTK81-luc vector (56).

{Delta}SRE plasmid contains the HMG CoA reductase promoter with a mutated SRE (5'-TGGCGGTG-3'). The oligonucleotide-directed mutagenesis was performed according to the method described by Osborne (5).

EMSAs
EMSAs were performed with synthetic oligonucleotides. In addition to the ERE-consensus and the Red-ERE oligonucleotides, the following oligonucleotides were used as competitors for EMSA: 5'-TCGAGTGCCTAGAGAACAAACTGTTCTGACTCAAC-3', encompassing a progesterone- responsive element (47), and 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' containing a cAMP-responsive element (17). An oligonucleotides containing the HMG CoA reductase promoter sequence from -111 to -72 was used for EMSA to detect complexes with ER and CREB. The complementary strands were annealed in equimolar amounts (10 nmol each) in 100 µl of annealing buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 30 mM KCl), by denaturation (5 min at 95 C) and cooling down to room temperature. Double-stranded oligonucleotides were radiolabeled using T4 polynucleotide kinase and [32P]-{gamma}-ATP. Nuclear extracts containing ER were prepared from MCF-7 cells treated for 2 h with 10 nM 17ß-estradiol, as previously described (57). Binding reactions were carried out in 30 µl reaction buffer containing 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 0.1 mM dithiothreitol, 10% glycerol, 5 µg BSA, 1 mM MgCl2, 90 mM NaCl, 0.1–0.5 ng radiolabeled DNA probe, and 2 µg calf thymus DNA. Specific competition assays were performed by adding 10- to 500-fold molar excess of unlabeled oligonucleotides. Five-microliter aliquots of the nuclear extract were added to the binding reaction and incubated for 30 min at room temperature. For the detection of retarded complexes, the reaction mixture was subjected to electrophoresis for 3 h on 4% polyacrylamide gels (acrylamide-bisacrylamide ratio, 30:1) at 11 V/cm in 0.5 x TBE buffer (Tris-borate-EDTA). Results were visualized by autoradiography of the dried gel and analyzed using a PhosphorImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). The monoclonal ER{alpha} antibody (C-314, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was included in the incubation mixture for supershift experiments.

Transfection Assays
MCF-7, Cos-1, Fto, and HepG2 cells were cultured in DMEM supplemented with 10% FCS. For transfection, 5 x 105 cells were plated on 60-mm plates. After 24 h, the medium was replaced by DMEM without phenol red supplemented with 10% charcoal-stripped FCS. Transfections were performed 48 h later using the diethylaminoethyl-dextran method (48) for MCF-7 cells and the calcium phosphate precipitation method (35) for Cos-1, Fto, and HepG2 cells. The amount of reporter plasmid used for each assay was 3 µg. For the experiments performed in Cos-1, Fto, and HepG2 cells, 0.2 µg of the expression vector for the human {alpha}-ER [pHEGO, (58)] was cotransfected. As an internal reference for transfection efficiency, 3 µg of the pRSV-lacZ plasmid were also cotransfected. After 24 h the medium was replaced by fresh medium containing the appropriate hormones or the corresponding solvent, and cells were cultured for additional 48 h. Cell extracts were prepared by three cycles of freezing and thawing. Protein concentration was measured by Bradford assay. CAT, luciferase, and ß-galactosidase activities were performed as described previously (31, 59).

Computer Analysis
Comparison and alignment of the different promoters were performed using the Wisconsin Package (Genetics Computer Group, Inc., Madison, WI) installed on a OpenVMS AXP station. Screening for the potential EREs was performed using MatInspector software (60).


   ACKNOWLEDGMENTS
 
We wish to thank P. Venditti, V. A. Raker, and J. Klug for the helpful discussions. We are grateful to A. Scholz for providing the recombinant ER and the p4xCRE vector and to A. E. Wakeling for the generous gift of antiestrogen ICI 164,384.


   FOOTNOTES
 
Address requests for reprints to: Miguel Beato, Institute for Molecular Biology, Phillips University, D-35033 Marburg, Germany.

L.D.C. was partially supported by a European Molecular Biology Organization short-term fellowship. This research was supported by a grants from the Deutsche Forschungsgemeinschaft, the Fonds der chemischen Industrie, and the European Union to M.B., and from MURST (40%–60%) to A.T.

Received for publication December 15, 1998. Revision received April 29, 1999. Accepted for publication May 18, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Rodwell VW, Nordstrom JL, Mitschelen JJ 1976 Regulation of HMG-CoA reductase. Adv Lipid Res 14:1–74[Medline]
  2. McGee TP, Cheng HH, Kumagai H, Omura S, Simoni RD 1996 Degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in endoplasmic reticulum membranes is accelerated as a result of increased susceptibility to proteolysis. J Biol Chem 271:25630–25638[Abstract/Free Full Text]
  3. Goldstein JL, Brown MS 1990 Regulation of the mevalonate pathway. Nature 343:425–430[CrossRef][Medline]
  4. Osborne TF, Gil G, Brown MS, Kowal RC, Goldstein JL 1987 Identification of promoter elements required for in vitrotranscription of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase gene. Proc Natl Acad Sci USA 84:3614–3618[Abstract/Free Full Text]
  5. Osborne TF 1991 Single nucleotide resolution of sterol regulatory region in promoter for 3-hydroxy-3-methylglutaryl coenzyme A reductase. J Biol Chem 266:13947–13951[Abstract/Free Full Text]
  6. Simonet WS, Ness GC 1989 Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA in rat liver. Glucocorticoids block the stabilization caused by thyroid hormones. J Biol Chem 264:569–573[Abstract/Free Full Text]
  7. Rosser DS, Ashby MN, Ellis JL, Edwards PA 1989 Coordinate regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and prenyltransferase synthesis but not degradation in HepG2 cells. J Biol Chem 264:12653–12656[Abstract/Free Full Text]
  8. Nakanishi M, Goldstein JL, Brown MS 1988 Multivalent control of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Mevalonate-derived product inhibits translation of mRNA and accelerates degradation of enzyme. J Biol Chem 263:8929–8937[Abstract/Free Full Text]
  9. Chambers CM, Ness GC 1997 Translational regulation of hepatic HMG-CoA reductase by dietary cholesterol. Biochem Biophys Res Commun 232:278–281[CrossRef][Medline]
  10. Gil G, Faust JR, Chin DJ, Goldstein JL, Brown MS 1985 Membrane-bound domain of HMG CoA reductase is required for sterol-enhanced degradation of the enzyme. Cell 41:249–258[CrossRef][Medline]
  11. Gillespie JG, Hardie DG 1992 Phosphorylation and inactivation of HMG-CoA reductase at the AMP-activated protein kinase site in response to fructose treatment of isolated rat hepatocytes. FEBS Lett 306:59–62[CrossRef][Medline]
  12. Osborne TF, Goldstein JL, Brown MS 1985 5' end of HMGCoA reductase gene contains sequences responsible for cholesterol-mediated inhibition of transcription. Cell 42:203–212[CrossRef][Medline]
  13. Smith JR, Osborne TF, Goldstein JL, Brown MS 1990 Identification of nucleotides responsible for enhancer activity of sterol regulatory element in low density lipoprotein receptor gene. J Biol Chem 265:2306–2310[Abstract/Free Full Text]
  14. Brown MS, Goldstein JL 1997 The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331–340[CrossRef][Medline]
  15. Yokoyama C, Wang X, Briggs MR, Admon A, Wu J, Hua X, Goldstein JL, Brown MS 1993 SREBP-1, a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene. Cell 75:187–197[CrossRef][Medline]
  16. Hua X, Yokoyama C, Wu J, Briggs MR, Brown MS, Goldstein JL, Wang X 1993 SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element. Proc Natl Acad Sci USA 90:11603–11607[Abstract/Free Full Text]
  17. Bifulco M, Perillo B, Saji M, Laezza C, Tedesco I, Kohn LD, Aloj SM 1995 Regulation of HMGCoA reductase gene expression in FRTL-5 cells. I. Identification and characterization of a cAMP-responsive element in the rat reductase promoter. J Biol Chem 270:15231–15236[Abstract/Free Full Text]
  18. Bruscalupi G, Castellano F, Scapin S, Trentalance A 1989 Cholesterol metabolism in frog (Rana esculenta) liver: seasonal and sex-related variations. Lipids 24:105–108[Medline]
  19. Luskey KL, Brown MS, Goldstein JL 1974 Stimulation of the synthesis of very low density lipoproteins in rooster liver by estradiol. J Biol Chem 249:5939–5947[Abstract/Free Full Text]
  20. Ma PTS, Yamamoto T, Goldstein JL, Brown MS 1986 Increased mRNA for low density lipoprotein receptor in livers of rabbits treated with 17{alpha}-ethinyl estradiol. Proc Natl Acad Sci USA 83:792–796[Abstract/Free Full Text]
  21. Di Croce L, Bruscalupi G, Trentalance A 1997 Independent responsiveness of frog liver low-density lipoprotein receptor and HMGCoA reductase to estrogen treatment. Pflügers Arch 435:107–111[CrossRef][Medline]
  22. Alonso MA, Fernandez de Quincoces A, Lacort M, Gandarias JM, Ochoa B 1993 Feeding status-related effect of 17ß-estradiol on liver HMGCoA reductase. Exp Clin Endocrinol 101:123–130[Medline]
  23. Erickson SK, Jaeckle S, Lear SR, Brady SM, Havel RJ 1989 Regulation of hepatic cholesterol and lipoprotein metabolism in ethinyl estradiol-treated rats. J Lipid Res 30:1763–1771[Abstract]
  24. Di Croce L, Bruscalupi G, Trentalance A 1996 Independent behaviour of rat liver LDL receptor and HMGCoA reductase under estrogen treatment. Biochem Biophys Res Commun 224:345–350[CrossRef][Medline]
  25. Cypriani B, Tabacik C, Descomps B 1988 Effect of estradiol and antiestrogens on cholesterol biosynthesis in hormone-dependent and -independent breast cancer cell lines. Biochim Biophys Acta 972:167–178[Medline]
  26. Baranao JL, Hammond JM 1986 FSH increases the synthesis and stores of cholesterol in porcine granulosa cells. Mol Cell Endocrinol 44:227–236[CrossRef][Medline]
  27. Wahli W, Metha E 1991 Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression. FASEB J 5:2243–2249[Abstract]
  28. Morley P, Whitfield JF, Vanderhyden BC, Tsang BK, JS 1992 A new, nongenomic estrogen action: the rapid release of intracellular calcium. Endocrinology 131:1305–1312[Abstract]
  29. Beato M 1989 Gene regulation by steroid hormones. Cell 56:335–344[CrossRef][Medline]
  30. Beato M, Herrlich P, Schutz G 1995 Steroid hormone receptors: many actors in search of a plot. Cell 83:85–89
  31. Klein-Hitpass L, Schorpp M, Wagner U, Ryffel GU 1986 An estrogen responsive element derived from the 5' flanking of the Xenopus vitellogenin A2 gene functions in transfected human cells. Cell 46:1053–1061[CrossRef][Medline]
  32. Kumar V, Chambon P 1988 The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell 55:145–156[CrossRef][Medline]
  33. Klock G, Strahle U, Schultz G 1987 Oestrogen and glucocorticoid responsive element are closely related but distinct. Nature 329:734–736[CrossRef][Medline]
  34. Scholz A, Truss M, Beato M 1998 Hormone induced recruitment of Sp1 in vivo mediates estrogen activation of the rabbit uteroglobin gene in endometrial epithelium. J Biol Chem 273:4360–4366[Abstract/Free Full Text]
  35. Pecci A, Scholz A, Pelster D, Beato M 1997 Progestins prevent apoptosis in a rat endometrial cell line and increase the ratio of bcl-XL to bcl-XS. J Biol Chem 272:11791–11798[Abstract/Free Full Text]
  36. Tam SP, Brissette L, Ramharack R, Deeley RG 1991 Differences between the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and low density lipoprotein receptor in human hepatoma cells and fibroblasts reside primarily at the translational and post-translational levels. J Biol Chem 266:16764–16773[Abstract/Free Full Text]
  37. Shimomura I, Shimano H, Horton JD, Goldstein JL, Brown MS 1997 Differential expression of exons 1a and 1c in mRNAs for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells. J Clin Invest 99:838–845[Medline]
  38. Vallett SM, Sanchez HB, Rosenfeld JM, Osborne TF 1996 A direct role for sterol regulatory element binding protein in activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene. J Biol Chem 271:12247–12253[Abstract/Free Full Text]
  39. Montminy MR, Gonzalez GA, Yamamoto KK 1990 Regulation of cAMP-inducible genes by CREB. Trends Neurosci 13:184–188[CrossRef][Medline]
  40. Inoue S, Orimo A, Hosoi T, Kondo S, Toyoshima H, Kondo T, Ikegami A, Ouchi Y, Orimo H, Muramatsu M 1993 Genomic binding-site cloning reveals an estrogen-responsive gene that encodes a RING finger protein. Proc Natl Acad Sci USA 90:11117–11121[Abstract/Free Full Text]
  41. Seiler-Tuyns A, Walker P, Martinez E, Merillat A, Givel F, Wahli W 1986 Identification of estrogen-responsive DNA sequences by transient expression experiments in a human breast cancer cell line. Nucleic Acids Res 14:8755–8770[Abstract/Free Full Text]
  42. Weisz A, Rosales R 1990 Identification of an estrogen response element upstream of the human c-fos gene that binds the estrogen receptor and the AP-1 transcription factor. Nucleic Acids Res 18:5097–5106[Abstract/Free Full Text]
  43. Wang F, Porter W, Xing W, Archer TK, Safe S 1997 Identification of a functional imperfect estrogen-responsive element in the 5'-promoter region of the human cathepsin D gene. Biochemistry 36:7793–7801[CrossRef][Medline]
  44. Nunez AM, Berry M, Imler JL, Chambon P 1989 The 5' flanking region of the pS2 gene contains a complex enhancer region responsive to oestrogens, epidermal growth factor, a tumour promoter (TPA), the c-Ha-ras oncoprotein and the c-jun protein. EMBO J 8:823–829[Medline]
  45. Slater EP, Redeuihl G, Theis K, Suske G, Beato M 1990 The uteroglobin promoter contains a noncanonical estrogen responsive element. Mol Endocrinol 4:604–610[Abstract]
  46. Cypriani B, Tabacik C, Descomps B, Crastes de Paulet A 1988 Role of estrogen receptors and antiestrogen binding sites in an early effect of antiestrogens, the inhibition of cholesterol biosynthesis. J Steroid Biochem 31:763–771[CrossRef][Medline]
  47. Chalepakis G, Schauer M, Cao XA, Beato M 1990 Efficient binding of glucocorticoid receptor to its responsive element requires a dimer and DNA flanking sequences. DNA Cell Biol 9:355–368[Medline]
  48. Truss M, Bartsch J, Schelbert A, Haché RJG, Beato M 1995 Hormone induces binding of receptors and transcription factors to a rearranged nucleosome on the MMTV promoter in vivo. EMBO J 14:1737–1751[Medline]
  49. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA, Rose DW, Glass CK et al. 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414[CrossRef][Medline]
  50. Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montminy MR, Goodman RH 1993 Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855–859[CrossRef][Medline]
  51. Oliner JD, Andresen JM, Hansen SK, Zhou S, Tjian R 1996 SREBP transcriptional activity is mediated through an interaction with the CREB-binding protein. Genes Dev 10:2903–2911[Abstract/Free Full Text]
  52. Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The trascriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87:953–959[CrossRef][Medline]
  53. Zhou Y, Watters JJ, Dorsa DM 1996 Estrogen rapidly induces the phosphorylation of the cAMP response element binding protein in rat brain. Endocrinology 137:2163–2166[Abstract]
  54. Rudling MJ 1987 Role of the liver for the receptor-mediated catabolism of low-density lipoprotein in the 17 {alpha}-ethinylestradiol-treated rat. Biochim Biophys Acta 919:175–180[Medline]
  55. Bertolotti M, Spady DK 1996 Effect of hypocholesterolemic doses of 17 {alpha}-ethinyl estradiol on cholesterol balance in liver and extrahepatic tissues. J Lipid Res 37:1812–1822[Abstract]
  56. Nordeen SK 1988 Luciferase reporter gene vectors for analysis of promoter and enhancer. Biotechniques 6:454–458[Medline]
  57. Andrews NC, Faller DV 1991 A rapid micropreparation techique for extraction of DNA binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 19:2499[Free Full Text]
  58. Kumar V, Green S, Staub A, Chambon P 1986 Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J 5:2231–2236[Medline]
  59. Ausubel FA, Brent R, Kingstone RE, Moore DD, Seidman JG, Smith JA, Struhl K 1996 Current Protocols in Molecular Biology. John Wiley & Sons, New York
  60. Quandt K, Frech K, Karas H, Wingender E, Werner T 1995 MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 23:4878–4884[Abstract/Free Full Text]



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