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Endocrinology Vol. 144, No. 7 2829-2835
Copyright © 2003 by The Endocrine Society

Expression of Functional Estrogen Receptor ß in Locus Coeruleus-Derived Cath.a Cells

Heather L. Rincavage, Donald P. McDonnell and Cynthia M. Kuhn

Department of Pharmacology and Cancer Biology and Program in Integrated Toxicology, Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: Dr. Cynthia Kuhn, Department of Pharmacology and Cancer Biology, Duke University Medical Center, P.O. Box 3813, Durham, North Carolina 27710. E-mail: ckuhn{at}duke.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen may have an important role in the brain beyond the development and regulation of reproductive function. Gender differences in the incidence of depression suggest that regulation of mood represents one such action. The locus coeruleus, a brain stem noradrenergic nucleus implicated in mood regulation, concentrates [3H]estradiol, but expression of the two estrogen receptor (ER) subtypes (ER{alpha} and ERß) varies across species. Further, the role of each subtype in estrogen action on noradrenergic neurons is unknown.

We examined the expression of ERs in the Cath.a (central-adrenergic-tyrosine-hydroxylase-expressing) cell line derived from mouse brain stem and found that they express ERß protein but not ER{alpha} protein. Transient transfection assays using an estrogen-responsive reporter gene indicate that ERß is functional. The pure estrogen antagonist ICI 182,780 completely abolished estrogen’s effects. Selective ER modulator results suggest that ER in Cath.a cells behaves in a manner consistent with ERß pharmacology. R,R-Tetrahydrochrysene, an ER{alpha} agonist, had no effect on luciferase-driven activity in Cath.a cells.

This study provides the first report of a cell line that spontaneously expresses functional ERß protein. Cath.a cells may prove to be a useful tool in elucidating basic pharmacologic properties of ERß. It may also help reveal the molecular mechanisms involved in mood regulation by estrogen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A GROWING BODY of evidence suggests that estrogen has an important role in the central nervous system beyond the development and regulation of reproductive function. In addition to being neuroprotective (1, 2), estrogen may affect mood, learning, and cognition (3, 4). These effects have stimulated interest in defining the role of estrogen in areas of the brain other than the hypothalamus, the area most relevant for understanding reproductive effects of estrogen. The locus coeruleus (LC) of rats, a noradrenergic cell nucleus in the brain stem that may be important in the regulation of mood (5), has been shown to concentrate [3H]estradiol (6, 7). Results from these early experiments suggested that this nucleus may contain specific estrogen receptors (ERs) and paved the way for future investigations. Estrogen acting within this brain stem region may have relevance to mood and other affective disorders, which disproportionately affect women (8, 9).

Conflicting findings about estrogen effects on this noradrenergic cell group have been published. A functional ER in the LC has been inferred by recent studies and suggests that estrogen has stimulatory effects on genes involved in norepinephrine synthesis. In the LC of ovariectomized female rat, estrogen treatment increases mRNA levels of tyrosine hydroxylase and dopamine ß-hydroxylase (10). Contradictory studies report that estrogen does not affect tyrosine hydroxylase gene expression in the rat LC (11). Yet other studies suggest an inhibitory effect of estrogen on norepinephrine neurons. Within the dorsolateral prefrontal cortex, ovariectomy of adult rhesus monkeys produces a net increase in the density of fibers immunoreactive for dopamine ß-hydroxylase. Estrogen replacement decreases immunoreactivity to normal levels (12).

These conflicting estrogenic effects might reflect target cell type and respective cellular components differences, as well as species differences in ER distribution. ER expression in other cell types, i.e. astrocytes, might also contribute to the estrogen effects observed in the intact brain (13, 14, 15). Differences in ER subtype distribution may also explain the disparities in estrogen action in different brain regions.

Two ERs have been identified, ER{alpha} and ERß (16), and each receptor has a distinct localization in the brain. The distribution of ER{alpha} and ERß mRNA in the rat central nervous system is well characterized (10, 17, 18, 19) and suggests that each receptor has a distinct localization (17, 19). The distribution of ER{alpha} protein has been investigated (20, 21). More recently, it has been shown that ERß mRNA is translated into immunoreactive protein throughout the rat brain, and immunoreactivity has been seen in the LC (17). The functional consequences of the receptors’ distinct localization are unknown.

Although ER{alpha} and ERß are structurally similar, the cellular response elicited by each in response to estrogen and other ligands may differ. In the absence of hormone, ERs are sequestered within the nuclei of target cells in a multiprotein inhibitory complex. The binding of hormone induces a conformational change within ERs, promoting dimerization and translocation to the nucleus. Once in the nucleus, ligand-bound receptors bind to DNA response elements [estrogen response elements (EREs)] located within the regulatory regions of target genes, ultimately altering target gene transcription and changing the phenotype of the target cell (22). ERs can also enhance or inhibit transcription by recruiting coactivator and corepressor proteins to the transcription complex (23). The relative expression of these coregulatory proteins may have a significant role in defining a cell’s estrogenic response.

An in vitro model of the LC with functional ERs would be an invaluable tool in which to study estrogen’s effects on the central noradrenergic system. We have demonstrated the expression of functional ERß, but not ER{alpha}, in the catecholamine-synthesizing Cath.a (central-adrenergic-tyrosine-hydroxylase-expressing) cells. Cath.a cells were derived from brain stem tumors of transgenic mice carrying the simian virus 40 T tumor antigen oncogene (24). Given the genotype of this cell line, it is most likely derived from the LC, a noradrenergic cell nucleus that projects to the forebrain, innervating the cortex, hippocampus, and hypothalamus (25). Cath.a cells offer a unique model for studying estrogen action on central monoamine systems for several reasons: 1) they are a well-characterized cell line possessing a neuronal phenotype; 2) they synthesize norepinephrine and all of the characteristic proteins of noradrenergic cells except the NE transporter; and 3) we have found that these cells express functional ERß, but not ER{alpha}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Cath.a cells (a generous gift from Dr. Dona Chikaraishi, Duke University, Durham, NC) were maintained in RPMI 1640 (Gibco, Invitrogen Corp., Carlsbad, CA) supplemented with 10% horse serum (Sigma, St. Louis, MO), 5% fetal calf serum (Hyclone Laboratories, Inc., Logan, UT), and 1% penicillin-streptomycin (Gibco, Invitrogen Corp.). MCF7 and HepG2 cells were maintained in MEM (Gibco, Invitrogen Corp.) supplemented 10% fetal calf serum (Hyclone Laboratories, Inc.), 0.1 mM sodium pyruvate, and 0.1 mM nonessential amino acids (Gibco, Invitrogen Corp.). For all experiments described below, cells were grown in the appropriate phenol red-free media, replacing 10% charcoal-dextran stripped fetal calf serum (Hyclone Laboratories, Inc.) for the usual serum(s) to avoid estrogenic effects and to up-regulate ER expression.

Hormone treatments
17ß-Estradiol was purchased from Research Biochemicals International (Natick, MA). Tamoxifen, 4-hydroxy tamoxifen (4OH tamoxifen), and clomiphene were purchased from Sigma. Raloxifene was a generous gift from E. Larson (Pfizer Pharmaceuticals, Groton, CT). The ER antagonist ICI 182,780 was purchased from Tocris Cookson (Ballwin, MO). R,R-Tetrahydrochrysene (R,R-THC) was a generous gift from Dr. John Katzenellenbogen (University of Illinois, Urbana-Champaign, IL). Stocks (1 mM) of all hormones were made in 100% ethanol and subsequently diluted to the appropriate concentrations in phenol red-free RPMI 1640 medium supplemented with 10% charcoal-dextran-stripped fetal calf serum.

Western blots
Cath.a cells were grown to confluency in 100 x 20-mm Petri dishes. Cells were lysed in 20 mM HEPES (pH 7.9), 20% glycerol, 400 mM KCl, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin. Whole-cell extracts (total protein 40 µg) were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel at 35 mA for 1 h. Protein was transferred to Immobilon membranes (Millipore Corp., Bedford, MA) at 200 mA for 1.5 h. Nonspecific binding was reduced by incubating the membranes for 1 h in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk. Blots were subsequently incubated with antisera specific for ERß (PAI-311, Affinity Bioreagents, Inc., Golden, CO; ABR, Inc., 1:1000) or ER{alpha} (H222, Dr. G. Greene, University of Chicago; 1:1000) diluted in the blocking buffer overnight at 4 C. Antibody-antigen complexes were detected with goat antirabbit (1:2000) conjugated to horseradish peroxidase (HRP) for ERß and antirat (1:2000) conjugated to HRP for ER{alpha}. Protein was visualized by an ECL detection system (Amersham Pharmacia Biotech, Arlington Heights, IL). Kaleidoscope markers were used to estimate protein size (Bio-Rad Laboratories, Inc., Hercules, CA). Baculovirus-expressed recombinant human ERß protein (53 kDa) was purchased from Pan Vera Corp. (Madison, WI). Blots were performed a minimum of three times for each ER.

Immunocytochemistry
Cath.a cells were plated into eight-well culture slides (Becton Dickinson and Co., Franklin Lakes, NJ) precoated with poly-D-lysine in white media 24 h before immunocytochemistry at a density sufficient to confer 80% confluency the following day. The following day, media were aspirated, and cells were fixed in fresh 4% paraformaldehyde for 3–5 min. Cells were washed three times for 5 min each time in PBS. Cells were washed in 0.5% H2O2 in PBS for 30 min to block endogenous peroxidase. Cells were incubated in blocking buffer (5% normal goat serum, 0.25% Triton X-100 in PBS) for 1 h. Primary antibodies diluted in blocking buffer were added to cells overnight at 4 C (PAI-310B or PAI-311 for ERß, ABR, Inc., 1:250; UCG62 or H222 for ER{alpha}, Dr. G. Greene, 1:1000). One well per slide always served as the negative control and did not receive primary antibody. Some experiments included wells in which secondary antibody treatment only was omitted. All other wells received either ER{alpha} or ERß antibodies. The following day, cells were washed three times for 5 min each wash in PBS supplemented with 0.1% Triton X-100 to help eliminate nonspecific antibody binding. Cells were then incubated in either goat antirabbit (Kirkegaard \|[amp ]\| Perry Laboratories, Gaithersburg, MD) or donkey antirat (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) biotinylated antibodies (1:2000 in PBS; 1 h, room temperature), washed again in PBS, then incubated in an avidin-biotin complexed HRP (Vector Laboratories, Burlingame, CA; 2 h, room temperature). Following several rinses in PBS, cells were reacted using diaminobenzidine/nickel as a chromagen (Vector Laboratories). Cells were dehydrated through increasing alcohol, coverslipped and mounted (Krystalon, Gibbstown, NJ). Slides were viewed and photographed with light microscopy. Each experiment was repeated a minimum of three times.

Transient transfection assays
For each experiment, cells were plated onto nine six-well plates coated with poly-D-lysine 24 h before transfection (5 x 10-5 cells per well), and DNA was introduced into cells using lipofectamine 2000 (Invitrogen). Triplicate transfections were performed using 18 µg total DNA. In standard transfections, 9000 ng of the reporter plasmid 3X-ERE-TATA-Luc, 600 ng of the pCMV-ß-gal normalization vector, and 8400 ng of the control plasmid pBSII-KS were used. The 3X-ERE-TATA-Luc reporter construct contains three copies of the vitellogenin A2 consensus sequence and a TATA box in front of the luciferase cDNA. Cells were washed with PBS, then incubated in the DNA/lipofectamine 2000 mix for 5 h. After 5 h, the DNA/lipofectamine 2000 mix was removed and replaced with phenol red-free RPMI medium supplemented with 10% charcoal-dextran-stripped fetal calf serum. The following day, three plates of cells were assayed for hormone responses by incubating with increasing concentrations (10 pM–100 nM) of one of four selective ER modulators (SERMs) (tamoxifen, 4OH tamoxifen, raloxifene, or clomiphene) or the estrogen antagonist ICI 182,780; another three plates of cells with increasing concentrations of 17ß-estradiol (10 pM–100 nM); and the last three plates of cells with increasing concentrations of 17ß-estradiol (10 pM–100 nM) combined with 1 µm of either ICI 182,780 or one of the four SERMs used in the first three plates. For experiments with R,R-THC, cells were plated onto nine six-well plates and transfected as described above. The following day, three plates of cells were assayed for a hormone response by incubating with increasing concentrations of 17ß-estradiol (10 pM–100 nM), another three plates with R,R-THC (10 pM–100 nM), and the last three plates with increasing concentrations of 17ß-estradiol (10 pM–100 nM) combined with 1 µm R,R-THC. Twenty-four hours later, luciferase activity was measured, and ß-galactosidase assays (26) were performed to normalize transfection efficiencies. Each experiment was replicated at least three times.

All data were analyzed using ANOVA with a P value set at 0.05. Post hoc analysis was performed on all significant effects using Fisher’s least significant difference (LSD) test (NCSS software, NCSS, Inc., Kaysville, UT).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cath.a cells express ERß but not ER{alpha} protein
To examine ER expression in Cath.a cells, we first performed a series of Western blots. We first compared ER{alpha} expression in Cath.a cells to ER{alpha} expression in MCF7 cells, which express ER{alpha} but not ERß, using a monoclonal antibody that is specific for ER{alpha} (H222). With our antibodies and techniques, we did not detect ERß in MCF7 cells. An ER{alpha}-immunoreactive protein migrating at 64 kDa was seen in MCF7 cells, as expected, but not in Cath.a cells (Fig. 1Go). In contrast, we detected an ERß-immunoreactive band in both Cath.a and PC12 cells that comigrated with baculovirus-expressed human ERß protein (Fig. 2Go). Derived from an adrenal medullary tumor, PC12 cells are reported to express ERß mRNA (27), but ERß protein has not been identified unequivocally in these cells. HepG2 cells, which express neither ER subtype, were included as a negative control and showed no immunoreactive ER as expected.



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  		Figure 1. Cath.a cells were immunoblotted for expression of ER{alpha} protein (H222, Dr. G. Greene, 1:1000). ER{alpha}-positive MCF7 cells were used as positive controls. ERß-negative HepG2 cells were used as negative controls. Molecular mass of standards (Bio-Rad) are indicated in kilodaltons.

 


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  		Figure 2. Cath.a and PC12 cells were immunoblotted for expression of ERß (PAI-311, Affinity Bioreagents, Inc.; 1:1000). Baculovirus-expressed recombinant human ERß protein was used as a positive control. ERß negative HepG2 cells were used as negative controls. Molecular mass of standards (Bio-Rad) are indicated in kilodaltons.

 
Next, we used immunocytochemistry to independently confirm the expression of ERß protein in Cath.a cells. Using the same antibodies that were used in the Western blots (H222 for ER{alpha} and PAI-311 for ERß), we were able to demonstrate that these cells only express ERß protein. Similar results were obtained using two different antibodies (UCG62 for ER{alpha} and PAI-310B for ERß) (Fig. 3Go). Incubation with antibodies for ER{alpha} produced no staining. In contrast, incubation with antibodies for ERß produced robust staining. Omission of primary antibodies produced no staining, indicating that the staining observed is ERß specific.



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  		Figure 3. Cath.a cells were immunostained for either ER{alpha} (H222 and UCG62) or ERß (PAI310B and PAI311). Omission of primary and secondary antibodies served as negative controls. A, No primary; B, no secondary; C, H222; 1:1000; D, UCG62, 1:1000; E, PAI-310B, 1:250; F, PAI-311, 1:250.

 
ERß is functional in Cath.a cells and is antagonized by the pure estrogen antagonist ICI 182,780 and by several selective ER modulators
To determine whether ERß in Cath.a cells was functional, we performed transient transfection assays of an estrogen-responsive reporter gene. First, we investigated the ability of 17ß-estradiol (10 pM–100 nM) to evoke a response in Cath.a cells. Our results (Fig. 4Go) show that estrogen treatment produced a concentration-dependent increase in transcriptional activity of the reporter gene, suggesting that the receptor is indeed functional. Next, we wanted to determine whether the receptor pharmacology is consistent with that reported for ERß. We tested the effects of increasing concentrations of 17ß-estradiol (10 pM–100 nM), increasing concentrations (10 pM–100 nM) of the pure estrogen antagonist ICI 182,780; and increasing concentrations of 17ß-estradiol (10 pM–100 nM) combined with 1 µm of ICI 182,780. Results are shown in Fig. 5Go.



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  		Figure 4. Cath.a cells were transiently transfected with the 3X-ERE-TATA-Luc reporter, the pCMV-ß-gal control plasmid, and pBSII-KS. After transfection, cells were treated with vehicle (nh) or increasing concentrations (ranging from 10-10–10-6 M of 17ß-estradiol for 24 h, and luciferase assays were performed. Each value was normalized to the ß-galactosidase activity. Each data point is the average of triplicate wells.

 


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  		Figure 5. Cath.a cells were transiently transfected with the 3X-ERE-TATA-Luc reporter, the pCMV-ß-gal control plasmid, and pBSII-KS. After transfection, cells were treated with vehicle (nh) or increasing concentrations (ranging from 10-11–10-7 M) of 17ß-estradiol, increasing concentrations of ICI 182,780, and increasing concentrations of 17ß-estradiol with 1 µM of ICI 182,780. Twenty-four hours later, luciferase assays were performed. Each value was normalized to the ß-galactosidase activity. Data shown are reported in percent max estradiol response. Each data point is the average of triplicate wells. All data were analyzed using ANOVA with a P value set at 0.05. Post hoc analysis was performed on all significant effects using Fisher’s LSD test. *, P < 0.05 as compared with the corresponding estradiol alone concentration.

 
In subsequent experiments, we tested the effects of increasing concentrations (10 pM–100 nM) of one of four SERMs (tamoxifen, 4OH tamoxifen, raloxifene, or clomiphene), increasing concentrations of 17ß-estradiol (10 pM–100 nM); and increasing concentrations of 17ß-estradiol (10 pM–100 nM) combined with 1 µm of the SERM being tested. Results from experiments using 4OH tamoxifen are shown in Fig. 6AGo; results from experiments using raloxifene are shown in Fig. 6BGo; results from experiments using tamoxifen are shown in Fig. 6CGo; and results from experiments using clomiphene are shown in Fig. 6DGo.



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  		Figure 6. Cath.a cells were transiently transfected with the 3X-ERE-TATA-Luc reporter, the pCMV-ß-gal control plasmid, and pBSII-KS. After transfection, cells were treated with (A) vehicle (nh) or increasing concentrations of 17ß-estradiol; increasing concentrations of 4OH tamoxifen; and increasing concentrations of 17ß-estradiol with 1µM 4OH tamoxifen. B, Vehicle (nh) or increasing concentrations of 17ß-estradiol; increasing concentrations of raloxifene. C, Vehicle (nh) or increasing concentrations of 17ß-estradiol; increasing concentrations of tamoxifen; and increasing concentrations of 17ß-estradiol with 1µM of tamoxifen. D, Vehicle (nh) or increasing concentrations of 17ß-estradiol; increasing concentrations of clomiphene; and increasing concentrations of 17ß-estradiol with 1µM of clomiphene. Twenty-four hours later, luciferase assays were performed. Each value was normalized to the ß-galactosidase activity. Data shown are reported in percent max estradiol response. Each data point is the average of triplicate wells. All data were analyzed using ANOVA with a P value set at 0.05. Post hoc analysis was performed on all significant effects using Fisher’s LSD test. *, P < 0.05 as compared with the corresponding estradiol alone concentration.

 
In all experiments, all compounds behaved as antagonists, decreasing or completely abolishing the transcriptional activity elicited by estrogen. None of the compounds exhibited partial agonist effects. The pure estrogen antagonist ICI 182,780 and 4OH tamoxifen completely blocked the effects of estrogen (100 pM–100 nM). Raloxifene decreased estrogen responsiveness, but not completely. Clomiphene decreased estrogen responsiveness, but the effects were overcome at higher doses of estradiol. Tamoxifen also decreased estrogen responsiveness at low concentrations only. The pharmacology of the compounds is consistent with our antibody data that indicate that Cath.a cells express functional ERß, but not ER{alpha}.

R,R-THC antagonizes ERß in Cath.a cells
We also tested the R,R-THC, an ER{alpha} agonist that functions as a complete antagonist on ERß (28). As shown in Fig. 7Go, R,R-THC did not significantly increase luciferase-driven activity at any of the concentrations tested. The addition of 1 µM R,R-THC to increasing concentrations of 17ß-estradiol was able to decrease estrogen responsiveness at several concentrations. These results our consistent with our previous findings that Cath.a cells do not express ER{alpha}, yet do express ERß.



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  		Figure 7. Cath.a cells were transiently transfected with the 3X-ERE-TATA-Luc reporter, the pCMV-ß-gal control plasmid, and pBSII-KS. After transfection, cells were treated with vehicle (nh) or increasing concentrations (ranging from 10-11–10-7 M) of 17ß-estradiol, increasing concentrations of R,R-THC, and increasing concentraions of 17ß-estradiol with 1 µM R,R-THC. Twenty-four hours later, luciferase assays were performed. Each value was normalized to the ß-galactosidase activity. Data shown are reported in percent max estradiol response. Each data point is the average of triplicate wells. All data were analyzed using ANOVA with a P value set at 0.05. Post hoc analysis was performed on all significant effects using Fisher’s LSD test. *, P < 0.05 as compared with the corresponding estradiol alone concentration.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report is the first that unequivocally demonstrates the spontaneous expression of functional ERß protein in a cell line. The expression of ERß mRNA has been reported in cell lines previously (27), but expression of mRNA does not always guarantee protein expression (29, 30, 31). Our findings are the first to show functional, spontaneously expressed receptor. Cath.a cells express functional ERß but do not express ER{alpha}. The presence of ERß protein was confirmed by Western blotting and immunocytochemistry, and reporter assays demonstrated that the receptor is functionally active. Western blot analyses of Cath.a whole-cell extracts demonstrated the presence of ERß, as indicated by a band comigrating with baculovirus-expressed ERß. The absence of ER{alpha} in Cath.a cells is suggested by the absence of a band corresponding to the 64-kDa band recognized by the monoclonal antibody H222 in ER{alpha}-positive MCF7 cells. Our findings that another catecholamine-synthesizing cell line, PC12 cells, express ERß protein were unexpected but is interesting because they are widely used as a model to study estrogen action on neuronal cell lines following transfection of ER{alpha} (27). The contribution of ERß may be significant in actions observed in these cells and may be important to take in consideration when studying estrogenic effects in these cells.

Immunocytochemistry experiments are concordant with those seen in the Western blots, suggesting that Cath.a cells express only ERß protein. These experiments also allow for the visualization of ERß, and clearly indicate that the staining is nuclear. The use of different antibodies with different epitopes than those used in our Western blotting experiments increases the validity of our findings, further supporting our findings that these cells express ERß. Specifically, PA1–311 recognizes an N-terminal epitope of ERß while PAI-301B recognizes a C-terminal epitope of ERß. In the same respect, UCG62 recognizes an N-terminal epitope of ER{alpha}, whereas H222 recognizes a C-terminal epitope of ER{alpha}.

Transient transfection assays showed the presence of functional ERß. A concentration-dependent increase in normalized luciferase output was observed in Cath.a cells exposed to increasing concentrations of estrogen. The effects of estrogen were effectively antagonized by the pure estrogen antagonist ICI 182,780 at concentrations of 100 pM and above. The SERMs clomiphene, raloxifene, tamoxifen, and 4OH tamoxifen also antagonized the effects of estrogen. Tamoxifen and clomiphene, which are triphenylethylenes, were the least effective at antagonizing estrogen in Cath.a cells. Raloxifene, a benzothiophene, was very effective at antagonizing estrogen. 4OH tamoxifen, a metabolite of tamoxifen, was the best antagonist. This greater effectiveness may be due to the altered stereochemistry that the hydroxyl group adds to the molecule, causing it to resemble a benzothiophene. The reason that one class of SERMs was a better antagonist than another in this system remains to be determined. Differing affinities for ERß may partly explain these effects. It is plausible that these observations reflect the relative expression of coactivators and/or corepressors and their role within Cath.a cells. Although coregulator expression was not determined in Cath.a cells, expression of SRC-1 mRNA in the LC has been previously demonstrated (32). None of the compounds exhibited partial agonist effects, which might have been the case had Cath.a cells expressed functional ER{alpha}.

The final compound tested in Cath.a cells, R,R-THC, is a novel, nonsteroidal ligand that shows ER subtype-selective differences in binding affinity and in transcriptional potency or efficacy. R,R-THC is an ER{alpha} agonist, but a complete antagonist on ERß. The reason for this pharmacologic specificity may reside in the binding properties of the compound; R,R-THC has a 3-fold preferential binding for ERß over ER{alpha} (28). R,R-THC had no effect on ERE-driven luciferase activity in Cath.a cells on its own, yet was able to suppress estradiol-stimulated transcriptional activity through ERß at several concentrations. These results are concordant with our other data, showing that Cath.a cells only express ERß.

It is interesting that the molecular pharmacology of these compounds is often studied in overexpression systems with ER{alpha} and/or ERß. It is possible that these compounds behave differently in these systems, and Cath.a cells may serve as an excellent model in which to compare these potential differences.

Most of the clinical research on SERMs focuses on their effects on bone resorption and reproductive health. However, some studies have examined their effects on depression, and results suggest that treatment with these drugs does negatively influence mood (33, 34, 35, 36). It is important to consider that many women on these therapies are experiencing other health problems (menopause, osteoporosis, subfertility), so studies need to be carefully designed to take these confounding variables into account. Our results suggest that SERMs might affect mood, given their ability to decrease or abolish estrogen-driven luciferase activity. Further investigation of this question may prompt the need for and development of SERMs that do not act in the brain.

The present findings raise the intriguing (although untested) possibility that ERß may be the ER expressed in the LC of mice, the source of Cath.a cells. Cath.a cells may prove to be tremendously useful for investigating the receptor’s role in regulating gene transcription in noradrenergic LC neurons and their projections. Because the LC is intimately involved in the control of mood, these findings imply that ERß may be important in mediating the effects of estrogen on mood. The LC has terminal fields within the hippocampus, cortex, spinal cord, cerebellum, and hypothalamus (25). These results suggest that ERß may mediate the effects of estrogen acting within the LC in these areas as well, although ER-expressing afferents to each these areas might make this speculation hard to test. ER expression in other cell types, i.e. astrocytes, might also contribute to the estrogen effects observed in the intact brain (13, 14, 15).

While both ER{alpha} and ERß mRNA have been identified in the primate LC (37, 38), the receptor protein complement in these cells is still unknown. Immunohistochemical studies performed before the discovery of ERß suggest that ER{alpha} is not expressed in primate LC (39). ERß expression in the LC has not yet been examined. Both receptor subtypes could be expressed in vivo. In fact, both receptor subtypes (10, 17, 18, 19, 40, 41) have been detected in rat LC. These findings suggest that interspecies variability and gender differences may exist in the expression of ER subtypes within the LC, which is a sexually dimorphic brain region (42). It is also possible that ER expression is heterogeneous throughout morphological subpopulations within the LC. Examination of ER protein expression throughout the mouse LC is needed to answer these questions. These studies are currently underway in the lab.

The other two major brain stem noradrenergic cell groups, the ventrolateral medulla and nucleus tractus solitarii, primarily express ER{alpha} in rodents, sheep, and primates (43, 44, 45). These neurons project to the hypothalamus and are involved in the regulation of GnRH secretion. These data suggest that ER{alpha} may be the important mediator of the effects of estrogen on reproductive function. Given the known differences in ER pharmacology, these observations imply that the mechanisms of gene regulation by estrogens probably differ across different brain regions. More complete characterizations of ER protein expression throughout the brain are needed to fully understand estrogenic effects in specific brain regions.

As a specific physiological role for ERß has yet to be determined, the discovery of a cell line that expresses only ERß may prove to be a useful tool in which to study estrogen’s role in the noradrenergic neurons of the LC, as well as elucidate basic pharmacologic properties of ERß. It may also help reveal the molecular mechanisms involved in mood regulation by estrogen.


   Acknowledgments
 
The authors give special thanks to members of the McDonnell lab for their helpful suggestions, generosity and friendship, and also to Dr. Dona Chikaraishi for her kind donation of the Cath.a and PC12 cells.


   Footnotes
 
This work was supported by National Research Service Award fellowship MH-65093 (to H.L.R.), by NIH Grants DK-48807 (to D.P.M.) and DA-02739 (to C.M.K.), and funds from R. J. Reynolds (to C.M.K.).

Abbreviations: Cath.a, Central-adrenergic-tyrosine-hydroxylase-expressing cell line; ER, estrogen receptor; ERE, estrogen response element; HRP, horseradish peroxidase; LC, locus coeruleus; LSD, least significant difference; 4OH tamoxifen, 4-hydroxy tamoxifen; R,R-THC, R,R-tetrahydrochrysene; SERM, selective ER modulator.

Received October 25, 2002.

Accepted for publication March 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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