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Endocrinology Vol. 141, No. 11 4056-4064
Copyright © 2000 by The Endocrine Society

ARTICLES

Differential Transcriptional Regulation of Rat Vasopressin Gene Expression by Estrogen Receptor {alpha} and ß1

Robert A. Shapiro1, Cong Xu1 and Daniel M. Dorsa1,2

Departments of Psychiatry and Behavioral Sciences
1 and Pharmacology
2 , University of Washington, Seattle, Washington 98195

Address all correspondence and requests for reprints to: Robert A. Shapiro, University of Washington, Psychiatry and Behavioral Sciences, Box 356560, Seattle, Washington 98195-6560. E-mail: shapiror{at}u.washington.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuronal expression of vasopressin messenger RNA (mRNA) and peptide has been shown to be estrogen dependent. A 5.5-kb genomic DNA fragment, 5' of the AVP coding region, was used in luciferase reporter assays to measure transcriptional activation by either estrogen receptor {alpha} or ß in response to various treatments. ER{alpha} and ERß displayed differential regulation of the AVP promoter. SK-N-SH cells transfected with ER{alpha} exhibited increased luciferase activity in response to estrogen, and the selective estrogen receptor modulators (SERMs), Tamoxifen, and ICI 182,780. Cells transfected with ERß exhibited a high constitutive activity, which is unchanged by exposure to SERMs but can be inhibited by estrogen. Deletion of 1.5 kb from the 5' end or mutation of a single estrogen response element (ERE)-like sequence resulted in loss of estrogen-dependent induction by ER{alpha} and increased the ability of estrogen to inhibit the high constitutive activity of ERß. The distal ERE-containing 1.5-kb fragment, when coupled to luciferase, is able to support both ER{alpha} and ERß mediated activation of transcription by estrogen. These results suggest that a single ERE in the distal 1.5-kb portion of the 5.5-kb fragment contains the primary positive estrogen responsive sequences for ER{alpha} and ERß. The data also suggest that sequences proximal to this element serve to inhibit transcription mediated by ERß.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ARGININE8-VASOPRESSIN (AVP) is a nonapeptide, which acts as a neurohypophyseal hormone involved in water metabolism and blood pressure regulation, and also as a CNS neurotransmitter or neuromodulator. The classical VP projections to the neurohypophysis arising from the magnocellular neurons of the supraoptic (SON) and the paraventricular (PVN) nuclei of the hypothalamus have been well described (1, 2). Extrahypothalamic expression of this gene has also been documented in the bed nucleus of the stria terminalis (BNST) and medial amygdala (MA) (3). Vasopressin secretion by those neurons into the septum and hippocampus has been implicated in learning and memory, affiliative and aggressive behavior (4, 5, 6, 7). Vasopressin gene transcription is positively regulated by osmotic stimulation, increases in cAMP and negatively regulated by glucocorticoids via the glucocorticoid receptor, both in vivo and in vitro (8, 9, 10).

In addition, VP is regulated by the steroids estrogen and testosterone. Numerous studies have demonstrated that gonadectomy dramatically reduces expression of AVP immunoreactivity and messenger RNA (mRNA) levels in both the BNST and MA (1, 11, 12, 13, 14, 15), which is restored in females by estrogen replacement and in males by testosterone. In contrast, AVP mRNA expression in the SON and the PVN remains unchanged (unpublished observation). The effects of estrogen are now known to be mediated by at least two different receptors termed ER{alpha} and ERß. These two appear to be colocalized with VP-producing cells in a region specific fashion. Estrogen receptor-{alpha} (ER{alpha}) mRNA is colocalized in vasopressenergic cells in the BNST, but no colocalization was observed in the SON or PVN (16). Interestingly, recent studies using sequences specific for the newly identified estrogen receptor ß (ERß) subtype (17, 18, 19) have colocalized ERß mRNA with vasopressinergic cells in the SON (16, 20, 21, 22), suggesting that changes in VP transcription may be differentially regulated by the two ER subtypes. In addition, both ER{alpha} and ERß have been colocalized in cells of the BNST, MA, and the periventricular preoptic nucleus (PE) (23).

The ERs have been shown to elicit gene transcription by interacting with at least two distinct promoter elements, the classical estrogen response element (ERE), which binds ligand-bound receptor dimers, and AP1 sites, which involves interaction with fos/jun complexes but does not require the DNA binding domain of the receptor (24). Both ER{alpha} and ERß activate classical EREs in response to E2 and the antiestrogens or selective estrogen receptor modulators (SERMs) Tamoxifen and ICI 182,780 can block this response (24). In contrast to their actions at EREs, distinct pharmacological profiles have been shown for the actions of ER{alpha} and ERß at AP1 sites (25). The SERMs, raloxifene, Tamoxifen, and ICI 182,780, are transcriptional activators of both ER{alpha} and ERß at these sites. In contrast, while E2 can activate ER{alpha} transcription at AP1 sites, it acts as an antagonist of SERM induced transcription by ERß. This model does not exclude the possibility of other regulatory elements being directly activated by SERMs (26). It is important to note that these studies used reporter constructs containing single response elements in nonneuronal cells (25). It has yet to be determined whether complex promoters containing multiple response elements will be regulated in a similar manner. In addition, ER{alpha}/ERß heterodimers have been shown to form in vitro (27, 28); however, their pharmacological profile has yet to be determined.

The studies reported here examine the estrogenic regulation of the complex AVP promoter, which contains several potential ERE and AP1-like sites, in SK-N-SH cells. SK-N-SH cells are a neuroblastoma cell line derived from a metastatic bone marrow tumor (29). They have been shown to express several neuropeptide receptors including µ and {delta}-opioid receptors (30), muscarinic acetylcholine receptors (31), as well as neurotransmitters and neurotransmitter transporters such as norepinephrine, dopamine, and their respective transporters (32). In addition, SK-N-SH cells exhibit neuropeptidergic properties in that they have been reported to express LH-releasing hormone (LHRH) (33).

To identify the promoter elements important in the estrogenic induction of the vasopressin gene we have compared the ability of ER{alpha} and ERß to activate a series of AVP promoter luciferase reporter constructs in response to estrogen and SERMs. Differential regulation of the AVP promoter by ER{alpha} and ERß was observed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
SK-N-SH cells, obtained from the ATCC (Manassas, VA), were cultured in Eagle’s MEM containing 10% FBS, 1% sodium pyruvate, nonessential amino acids, and penicillin-streptomycin. Cells were passaged once per week and were not used after ten passages. Forty-eight hours before transfection, the medium was changed to phenol-free MEM containing 10% charcoal stripped FBS (Sigma). MCF-7 cells, obtained from the ATCC, were cultured in Eagle’s MEM containing 10% FBS, bovine insulin (10 µg/ml), 1% sodium pyruvate, nonessential amino acids and penicillin-streptomycin.

Plasmids
The complementary DNAs (cDNAs) encoding both human HEO\ER{alpha} (34), and rat ERß (17) were subcloned into the expression vector, pcDNA3 (Invitrogen). A 5.5-kb fragment containing the rat vasopressin promoter (35) was excised from the RVP-Sal plasmid (generously provided by Dr. David Murphy, University of Bristol, UK) with Not1 and Sal1 and cloned into the Not1 and Xho1 sites of the luciferase reporter plasmid, pGL2-B (Promega Corp.). The resulting plasmid, (p5.5luc) was used to generate all other constructs.

The G400V mutation present in the original ER{alpha} clone (HEO) has been reported to effect the basal activity of ER{alpha} (36). To address the possibility that the differences in basal activity we have observed between ER{alpha} (HEO) and ERß are due to the G400V mutation, control experiments were performed comparing the activity of HEO (which contains a valine at amino acid 400) and HEGO (a glycine at amino acid 400) and ERß on p5.5luc. While HEGO does display a slightly higher basal activity than HEO, in no case did it approach the high, uninduced basal activity of ERß. Furthermore, both HEO and HEGO exhibit the same level estrogen-dependant activation (fold increase) of p5.5luc (data not shown).

Transient transfections
SK-N-SH cells (~5 x 105 cells) were transfected in six-well plates with 2 µg/well of each luciferase reporter and either human ER{alpha}/hEO plasmid (generously provided by Dr. Pierre Chambon, Strasbourg, France) or rat ERß (generously provided by Dr. George Kuiper, Sweden) for 7 h using standard calcium phosphate procedures described elsewhere (Promega Corp.). One microgram per well of pCH110, a ß-galactosidase reporter (Amersham Pharmacia Biotech, Uppsala, Sweden) was used for normalizing transfection efficiency.

Treatments
After transfection, the media were replaced with phenol red-free media supplemented 10% charcoal-stripped calf serum and allowed to recover overnight. Cells were treated in triplicate with various doses of water soluble 17ß-estradiol (Sigma), 1 µM Tamoxifen (Research Biochemicals International, Natick, MA), or 1 µM ICI 182,780 (Zeneca Pharmaceuticals LTD, London, UK) for 8 h and harvested in 0.25 ml of reporter lysis buffer (Promega Corp.). For luciferase assays, 100 µl of cell extract was mixed with luciferase assay reagent (Promega Corp.) according to the manufacturer’s protocol and read on an E.G.&G LB 953 AutoLumat luminometer for 30 sec/sample as previously described (37). For ß-galactosidase assays, 50 µl of cell extract was mixed with 50 µl of Assay 2x buffer (Promega Corp.), incubated at 37 C for 2 h and absorbance at 420 nM determined on a Packard Spectracount 96-well plate reader as previously described (37). Data are presented as a ratio of luciferase to ß-galactosidase activity.

Statistics
Statistical analysis was performed using the ANOVA Scheffé’s F test for posthoc significance.

Gel shift assay
Nuclear extracts were prepared from cultured MCF-7 cells as described (38, 39). Briefly, protein extracts (10 µg) were incubated with 1 µg poly(dI-dC) in 20 µl buffer [10 mM HEPES (pH 7.9), 1 mM dithiothreitol, 80 mM KCl, 2 mM MgCl2, 10% glycerol, and 3 µg/ml BSA] plus 32P 5'-end-labeled oligonucleotides (20,000 cpm) for 15 min at room temperature (40) with or without 100-fold excess of specific or nonspecific competitor. Complexes were resolved by electrophoresis at 4 C on a 6% nondenaturing acrylamide gel equilibrated in 0.25x TBE (5 mM Tris and 0.5 mM EDTA), dried and exposed to film for 2–3 days. Specific oligonucleotides used: ERE-1: 5'-AGTGGGCAGCCTCACCCCTA-3'; ERE-2: 5'-GCAGGGCCAGCCTGACCGTGT-3'; c-ERE: 5'-GATCCAGGTCACTGTGACCTG.

Mutagenesis
The distal 1.5-kb fragment (p5'-1.5luc) was excised from p5.5luc with Not1 and BglII and cloned into the Not1 and BglII site of pGL2-B. p4.0luc was generated by linearizing p5.5luc with Not1, partially digesting with BglII, filling in the ends with Klenow and religating the plasmid. The 1.1 kb proximal portion of the promoter was amplified by PCR from p5.5luc, cloned into TAPCR (Invitrogen), excised with Xho1 and HindIII and cloned into the Xho1 and HindIII sites of pGL2-B (p1.1luc).

Oligonucleotide-directed mutagenesis
Two point mutations were introduced into p5.5luc by Sequence Overlap Extension procedure (41). One mutation (pcon5.5luc) changes ERE-2 to a consensus ERE, the other (pmut5.5luc) changes two nucleotides in ERE-2 which disrupt the 5 base palindrome. The two changes have each been previously shown to functionally disrupt estrogen regulation of an ERE reporter gene in transient transfection experiments (42, 43). Overlapping PCR fragments were first generated in separate reactions and then reannealed and extended in a second PCR. In the first step, two oligonucleotides (complement and reverse complement) containing the desired nucleotide changes were synthesized for each mutation. Two fragments were then synthesized by PCR using a 5'oligo (5'-CATACatgcatGGACGCAC-3') and the reverse complement to generate the 5' fragment and with the complement (see below) and a 3' oligonucleotide (5'AGCAcccgggAGACAGAGTC-3') to generate the 3' fragment (95 C for 45 sec; 55 C for 60 sec; 72 C for 90 sec; 35 cycles). After gel purification, 10 ng of each fragment were mixed and used as template in a second PCR with the two outer oligonucleotides. The resulting 800-bp fragment was digested with Nsi1 and Sma1 and used to replace the corresponding fragment in 5.5luc. All nucleotide changes were confirmed by DNA sequencing. Oligonucleotides used are shown below. Mutated nucleotides in the ERE palindrome (underlined) are in bold type.

pmut5.5luc: 5'-AGCGCCAGCCTGGCCGTGTGTGCCAGAAGATCTTC-3'.

pcon5.5luc: 5'-GCTTCTGCAGGGTCAGCCTGACCGT-3'

DNA sequencing
Sequencing of the 5.5-kb promoter fragment was performed at the University of Washington Genome Center by fluorescent dye termination on a PE-ABI 373 using the ABI Prism Dye primer cycle sequencing kit and custom designed primers according to the standard protocol recommended by the manufacturer. The complete sequence has been deposited in GenBank (Accession No. AF112362).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A 5.5-kb genomic DNA fragment upstream of the rat vasopressin gene was cloned into a luciferase expression vector (p5.5luc, Fig. 1Go). SK-N-SH cells were then transiently transfected with plasmids encoding either human ER{alpha} (HEO) or rat ERß to examine the role of estrogen in mediating expression of the vasopressin gene.



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  		Figure 1. Structure of the rat AVP promoter and mutations used in cotransfection assays. 5.5 kb of the rat vasopressin promoter (GenBank Accession No. AF112362) was fused to the gene encoding luciferase (black striped box). Regions deleted are indicated by dashed line. Restriction sites (Not1 and BglII) marking the boundaries of p5'1.5luc are are indicated Potential ERE-1 (gray box), potential AP-1 sites white striped boxes. The sequence of ERE-2 (black box) and the point mutations introduced are compared with a consensus ERE sequence. Differences from the consensus sequence are underlined. M and C refer to constructs containing either a mutated (M) or a consensus (C) ERE.

 
Increasing amounts of either ER{alpha} (Fig. 2AGo) or ERß (Fig. 2BGo) were transfected with p5.5luc and treated with 50 nM 17ß-estradiol (E2). ER{alpha} transfected cells exhibited a plasmid dependant increase in E2-induced promoter activity. ERß transfected cells exhibited a marked receptor-dependant increase in constitutive activity. No significant activity was observed in the absence of transfected receptor. At low ERß concentrations, addition of E2 increased promoter activity, however, at high receptor concentrations (>=2 µg/well), ERß mediated transcription is inhibited by E2. No receptor-dependent increases in luciferase expression were observed in cells transfected with the control luciferase plasmid, pGL2b (Fig. 2CGo). In fact, a small decrease was observed.



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  		Figure 2. Both ER{alpha} and ERß can activate the AVP promoter in vitro. SK-N-SH cells were transiently transfected with increasing amounts of plasmids encoding either ER{alpha} (A) or ERß (B), p5.5luc and the pCH110-ß-galactosidase reference plasmid. Cells were treated with vehicle (open) or 50 nM 17ß-estradiol (solid) for 7–8 h. Data shown are representative of three experiments performed in triplicate. (C) Cells were transiently transfected with 2 µg of ER{alpha} or ERß and either pGL2b (promoterless vector) or p5.5luc. Cells were treated with vehicle, 17ß-estradiol (50 nM) or ICI 182,780 (200 nM). Data shown are representative of two experiments performed in triplicate. *, Significant (P < 0.05) differences between vehicle and drug treatments.

 
We next examined the ability of increasing amounts of E2, ICI 182,780 and Tamoxifen to activate p5.5luc when cotransfected with 2 µg of either ER{alpha} or ERß. As observed in Fig. 2AGo, E2 increased ER{alpha} mediated transcription while E2 inhibited the high constitutive activity of ERß (Fig. 3AGo). Both ICI and Tamoxifen also demonstrated agonist activity in ER{alpha} transfected cells, an effect consistent with action at AP1-like elements of the AVP promoter but had no effect on the constitutive activity of ERß (Fig. 3Go, B and C). No ligand-induced increase in luciferase activity was observed in cells transfected with the control plasmid, pcDNA3.1 (data not shown). Thus, in contrast to ER{alpha}, which is able to activate the AVP promoter in response to E2 and SERMs, transfection with ERß results in a high transcriptional activity with only a minimal response to added ligands.



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  		Figure 3. Estrogen, Tamoxifen and ICI 182,780 increase transcription by ER{alpha} but not by ERß. Cells were transfected with 2.0 µg of plasmid encoding either ER{alpha} (open) or ERß (solid) as described and treated with increasing amounts of (A) 17ß-estradiol, (B) ICI 182,780, or (C) Tamoxifen as indicated. Data shown are representative of three experiments performed in triplicate. *, Significant (P < 0.05) differences between vehicle and drug treatments.

 
To localize regions of the promoter required for the ER{alpha} and ERß-mediated induction by estrogen, deletion mutations were constructed (p4.0luc and p1.1luc), fused to the luciferase gene (Fig. 1Go) and tested for estrogen-mediated luciferase activity (Fig. 4Go). At receptor concentrations (1 µg) that exhibit E2 stimulation for both ER{alpha} and ERß (see Fig. 2BGo), deletion of the distal 1.5 kb (p4.0luc) resulted in a complete loss of E2 activation when cotransfected with ER{alpha} (Fig. 4aGo). In fact, a small but significant inhibition is observed, indicating that the critical sequences for ER{alpha} activation by E2 reside within a 1.5 kb fragment located 4.0 kb upstream of the AVP coding region. Deletion of an additional 3.0 kb (p1.1luc) resulted in the loss of significant inhibition by estrogen, suggesting the presence of inhibitory sequences within the 3.0-kb segment.



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  		Figure 4. Mutation of a distal ERE abolishes estrogen-induced transcription of the AVP promoter. Cells were transfected as described with the indicated promoter constructs and 1 µg of plasmid encoding either ER{alpha} (A) or ERß (B). ER{alpha} activation of p5.5luc was included in B as an additional positive control for activation. Cells were treated with vehicle (open), 10 nM (hatched) or 50 nM 17ß-estradiol (solid) and analyzed for luciferase activity as described. Data shown are representative of three experiments performed in triplicate. *, Significant (P < 0.05) differences between vehicle and drug treatments.

 
In cells transfected with 1 µg of ERß (Fig. 4BGo), deletion of the distal 1.5 kb (p4.0luc) increased the level of constitutive activity, and resulted in E2-induced inhibition. Further deletion (p1.1luc) reduced the constitutive activity but retained significant inhibition. These data suggest that sequences located in the 1.1-kb portion of the promoter are responsible for the high constitutive activity of ERß in the absence of E2 and the inhibition of transcription in response to E2.

To confirm that sequences within the 5' 1.5 kb fragment were capable of mediating E2 activation by ER{alpha}, p5'-1.5luc was tested with both ER{alpha} and ERß for estrogen-mediated luciferase activity (Fig. 5AGo). E2 increased luciferase activity in cells expressing either ER{alpha} or ERß (1 µg), confirming the importance of these sequences in mediating E2 activation of the AVP gene. Cells transfected with ERß and p5'-1.5luc responded more robustly to E2 when compared with the 5.5-kb construct, possibly due to a lower constitutive activity (Fig. 5AGo). Removal of the proximal 4.0 kb dramatically altered the effects of SERMs on promoter activation by the two receptor subtypes. In ER{alpha} transfected cells, ICI was able to inhibit E2 activation to the same level as ICI alone. Tamoxifen was less potent than E2, but its effect in combination with E2 was additive (Fig. 5BGo). Both ICI and Tamoxifen antagonized activation of ERß by E2 but had little agonist activity of their own (Fig. 5CGo), consistent with a classical ERE response. These results suggest that the sequences required for SERM-mediated transcription of VP by ER{alpha} and the high constitutive activity of ERß reside primarily in the proximal 4.0-kb region of the promoter, whereas E2-mediated transcription requires the 1.5-kb distal region.



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  		Figure 5. Both ER{alpha} and ERß activate a distal segment of the AVP promoter. A, Cells were transiently transfected with p5.5luc or p5'-1.5luc, and plasmids encoding 1 µg of either ER{alpha} or ERß. Data shown are representative of three experiments performed in triplicate. Cells cotransfected with p5'-1.5luc and either ER{alpha} (B) or ERß (C) were treated vehicle (V), 17ß estradiol (E), ICI (I) or Tamoxifen (T) as indicated. Data shown are the average of three experiments performed in triplicate. *, Significant (P < 0.05) differences between vehicle and drug treatments.

 
The sequence of the entire 5.5-kb promoter fragment was determined (submitted to GenBank (Accession No. AF112362). Sequence analysis of the 1.5-kb distal region revealed two potential EREs, ERE-1 and ERE-2 (Fig. 1Go). Oligonucleotides containing both sequences were synthesized and tested in gel mobility shift assays with nuclear extracts of MCF7 carcinoma cells, which contain ER{alpha} (44, 45). Only one sequence (ERE-2) demonstrated specific binding (Fig 6Go). This ERE sequence was inactivated (pmut5.5luc) by site-directed mutagenesis (Fig. 1Go) and tested for luciferase activity in a cotransfection assay with 1 µg of ER{alpha} (Figs. 4AGo and 7AGo) or ERß (Figs. 4BGo and 7BGo).



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  		Figure 6. ERE-2 but not ERE-1 can specifically bind nuclear extracts from MCF-7 cells. Radiolabeled oligonucleotides containing the two potential EREs found in p5'1.5luc were compared with a consensus ERE (cERE) in a gel mobility assay. Ten micrograms of nuclear extracts from MCF7 cells were incubated with each oligo (-) in the presence of specific (sp) and nonspecific (nsp) unlabeled oligonucleotides and separated by PAGE. Arrow indicates the location of the shifted band. Figure is representative of three independent experiments.

 


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  		Figure 7. Mutations in the distal promoter affect estrogen-mediated but not SERM-mediated transcription. Cells were cotransfected with either p4.0luc, p5.5luc, or pmut 5.5luc and plasmids encoding 1 µg of either ER{alpha} (A) or ERß (B) as described in Fig. 2Go. Cells were treated as indicated with vehicle (open), 10 nM 17ß-estradiol (right diagonal), 1 µM ICI 182,780 (solid), or 1 µM Tamoxifen (left diagonal). Data shown are the average of three experiments performed in triplicate. *, Significant (P < 0.05) differences between vehicle and drug treatments.

 
As previously observed with p4.0luc, which deletes 1.5 kb containing ERE-2, cells cotransfected with ER{alpha} and pmut5.5luc showed no activation by E2 (Figs. 4AGo and 7AGo) confirming the requirement for this distal ERE in mediating ER{alpha} activation by E2. Mutation of the same ERE to a consensus ERE (pcon5.5luc, Fig. 1Go) resulted in increased promoter activity compared with p5.5luc (Fig. 4AGo). The mutation had no effect on the ability of Tamoxifen or ICI to activate the promoter (Fig. 7AGo), further suggesting that SERM activation is not ERE mediated, but rather by sequences within the proximal 4.0-kb region. Indeed, sequence analysis of this region of the promoter revealed several potential AP-1 like sequences.

Cells transfected with ERß and pmut5.5luc maintained the high constitutive activity previously observed with p5.5luc and p4.0luc. In addition, E2 inhibited luciferase activity to the same extent as was previously observed using the p4.0luc construct (Figs. 4BGo and 7BGo). In the same context, the consensus ERE exhibited increased luciferase activity when compared with either p5.5luc or p5.5luc cotransfected with ER{alpha}. Thus, the distal ERE provides positive ERß-mediated regulation by E2. When the ERE is removed, either by deletion or point mutation, E2 treatment results in ERß-mediated inhibition of AVP transcription. Similar to the results observed with ER{alpha}, the mutation had no effect on the ability of Tamoxifen or ICI to activate the promoter in ERß transfected cells (Fig. 7BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified an ERE in the Rat AVP promoter, which is capable of mediating estrogenic induction of AVP expression. Sequence analysis of the 5.5-kb promoter region identified several other possible transcriptional regulatory elements including several AP1 and CRE sites. Because both ER subtypes have been shown to be active at multiple transcriptional promoter elements, it is important to understand how ER{alpha} and ERß might interact with these various elements to give rise to the complex regulation observed in vivo. In this report, we provide evidence for the differential regulation of AVP transcription by ER{alpha} and ERß in a neuronal cell line.

ER{alpha} activates the AVP promoter in response to E2 by using the distal ERE element in a manner consistent with the classical model of estrogen receptor activation. Mutations that remove the ERE lose the ability to respond to E2 but have no effect on the agonist activity of Tamoxifen or ICI 182,780, demonstrating that SERM activation of the VP promoter does not require an ERE element. While the data (Fig. 4AGo) suggests the possible presence of inhibitory sequences for ER{alpha} within the p4.0luc construct, which can be removed by further deletion (p1.1luc), the inhibition was not consistently observed in all experiments (Fig. 7AGo). This inconsistency may be due to variation in the expression of unknown nuclear coactivator proteins that maybe involved. The enhancement of transcription by Tamoxifen and ICI 182,780 indicates that elements located elsewhere in the promoter are also major contributors to transcriptional activation by ER{alpha}. Agonist effects of Tamoxifen and ICI 182,780 mediated by ER{alpha} have been reported for AP1 sites (24) and also cAMP and CRE mediated gene transcriptional effects (37). Iwasaki et al. (10) have described a proximal CRE-like element capable of supporting this mode of regulation. It is thought that cAMP-dependent regulation of the gene is central to the osmotic regulation of magnocellular AVP gene transcription (46).

Cells transfected with ERß exhibit a more complex transcriptional profile. At low levels of transfected ERß (Fig. 2BGo), E2 is able to induce expression of luciferase from the full-length promoter. Transfection with increasing amounts of ERß results in high constitutive activity that is inhibited by E2 (Figs. 2BGo and 3AGo) and unaffected by the addition of SERMs. At low receptor levels (1 µg), constructs that delete or mutate the distal ERE maintain the constitutive activity, but are inhibited by E2, suggesting that the constitutive activity may be due to transcription from AP1 like sequences (25) present within the 4.0-kb construct. When cotransfected with the 1.5 kb distal promoter, which contains the ERE element, the constitutive activity is decreased and E2 increases ERß-mediated transcription. Thus, in cells expressing low levels ERß, the opposing effects of E2 stimulation at the ERE and inhibition at AP1-like sequences results in relatively constant vasopressin transcription.

Cells transfected with ERß and the pGL2b plasmid vector occasionally showed small but highly variable increases in luciferase activity. In contrast, cells transfected with ERß (1 µg) and p5.5luc consistently increased luciferase activity. Given the magnitude of ERß-mediated luciferase expression, we were initially concerned that these effects might be derived from sequences in the luciferase vector backbone. Vector sequences have been reported to confound the interpretation of data from promoter studies due to either position effects of enhancer-like sequences or the presence of promoter elements contained within the vector sequences (47). Sequence analysis of the pGL2b vector failed to reveal either ERE or AP-1 like sequences. The strongest argument against the involvement of vector sequences is that both point mutations within the ERE, which do not change the length of the promoter, and 1.5-kb deletion of the ERE (which does change the length of the promoter) are able to abolish the increase in transcription observed in both ER{alpha} and ERß transfected cells.

These observations suggest a model for the differential regulation of AVP transcription by ER{alpha} and ERß. According to this model, cells expressing ER{alpha} could respond to E2 by increasing VP mRNA transcription, while in cells expressing ERß, the opposing actions of E2 on ERE and AP1-like elements would result in relatively stable AVP mRNA levels. In addition, these results suggest that SERMs may activate vasopressin expression in cells expressing ER{alpha}.

The physiological relevance of this model is demonstrated by the regulation of VP gene expression by hormones in vivo. Vasopressin is expressed in the PVN, SON, BNST, and the MA. It has been previously shown that gonadectomy virtually abolishes VP mRNA and peptide expression in the BNST, and this effect can be reversed by local implants (3) of testosterone or estrogen. The post castration decline of VP mRNA in the BNST is not rapid (3–7 days), and interestingly, the peptide remains detectable for up to 8 weeks (13). While we have previously provided evidence of a transcriptional effect measured by intronic probe detection of VP primary transcript in situ hybridization studies (48), others have suggested that changes in mRNA poly(A)-metabolism may also play a role (49). Consistent with the loss of VP mRNA, is the fact that both ER{alpha} and ERß are highly expressed in the BNST and the MA. The mix of ER{alpha} and ERß might explain the slow decline of VP mRNA and peptide levels in these regions. In cells expressing ER{alpha}, estrogen deficiency would tend to reduce transcription, but in cells expressing ERß the higher constitutive activity may tend to oppose this effect. Because cells in the BNST, MA, and PE have been recently shown to express both receptor subtypes (23), it will be interesting to determine the effect of E2 on ER{alpha}/ERß heterodimers on VP mRNA expression. Most significantly, relatively little change in VP mRNA is observed in the SON and PVN, regions that express exclusively ERß (16, 21, 22). Thus, the opposing effects of E2 at ERE and AP1 sites could explain the relative lack of change in AVP mRNA in these neurons.

In summary, Paech et al. (25), have recently described differential ligand activation of ER{alpha} and ERß at AP1 sites. In their model, using a consensus AP1 reporter construct derived from the collagenase promoter, estrogen had no effect on ERß activation of AP1 sites but was able to antagonize SERM activation. In the present analysis of the AVP gene, we are able to test this model on a complex, natural promoter. We report a constitutive activity of ERß and observe estrogen acting as an inverse agonist in cells expressing relatively high levels of ERß or in our ERE-deficient constructs. A recent report by Forman et al. (50) describes a similar example of inverse agonism by androstane metabolites of the constitutive androstane receptor-ß (CAR-ß), another member of the nuclear steroid receptor family. Although no increased constitutive activity by ERß at AP1 sites was reported by Paech et al. (25), examination of the results presented suggests a small inhibitory effect of E2. This may reflect differences in the ability of ERß to activate a single AP1 reporter construct as opposed to a complex promoter containing multiple AP-like sequences. Alternatively, the presence of AP1-like variants may be preferentially activated in the presence of ERß. It has also been reported (24, 25) that ER{alpha} is able to activate transcription at AP1 elements in response to E2. In the AVP promoter, no E2-mediated transcription was observed in constructs that removed the distal ERE even though there are several AP1-like elements present that should be able to mediate SERM activation of the promoter. Nevertheless, our results are consistent with the model proposed by Paech et al. (25) in that estrogen can negatively regulate nonERE transcription by ERß. Experiments are in progress to identify those sequences responsible for the high constitutive activity of ERß and the negative regulation by E2.


   Acknowledgments
 
The authors wish to thank David Murphy of the University of Bristol for the genomic clone containing the rat vasopressin promoter, Pierre Chambon (Strasbourg, France) for use of the human ER{alpha} cDNA clones HEO and HEGO, and George Kuiper (Karolinska Institute, Sweden) for the Rat ERß cDNA, Zeneca Pharmaceuticals for the gift of ICI 182,780, Ms. Zhou Yang for technical assistance, Pam McMillan Ph.D. for critical review of the manuscript and Sherry Neher for help with the preparation of this manuscript.


   Footnotes
 
1 This work was supported by Public Health Service Grant NS20311 and the Alzheimer’s Disease Research Center of the University of Washington, AG-05136. These sequence data have been submitted to the DNA Database of Japan/European Molecular Biology Laboratory/GenBank databases under accession number AF112362. Back

Received May 23, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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