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Molecular Endocrinology, doi:10.1210/me.2006-0027
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Molecular Endocrinology 20 (9): 2080-2092
Copyright © 2006 by The Endocrine Society

Coordinate Regulation of Neuropeptide Y and Agouti-Related Peptide Gene Expression by Estrogen Depends on the Ratio of Estrogen Receptor (ER) {alpha} to ERß in Clonal Hypothalamic Neurons

Danny Titolo, Fang Cai and Denise D. Belsham

Departments of Physiology (D.T., F.C., D.D.B.), Obstetrics and Gynaecology (D.D.B.), and Medicine (D.D.B.), University of Toronto and Division of Cellular and Molecular Biology (D.D.B.), Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: Denise D. Belsham, Ph.D., Department of Physiology, University of Toronto, Medical Sciences Building 3247A, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: d.belsham{at}utoronto.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Neuropeptide Y (NPY) and agouti-related peptide (AgRP) stimulate feeding, whereas NPY also facilitates the estrogen-mediated preovulatory GnRH surge. In addition to regulating reproductive function, estrogen also acts as an anorexigenic hormone, although it is not yet known which hypothalamic neurons are involved in this process. We hypothesize that estrogen may directly control hypothalamic NPY and/or AgRP synthesis to influence energy homeostasis. Using two clonal, murine hypothalamic neuronal cell models, N-38 and N-42, we demonstrate that 17ß-estradiol differentially regulates estrogen receptor (ER){alpha} and ERß levels, as well as NPY and AgRP gene expression in a manner that is temporally coordinated with the changes in ER abundance. The estrogen-mediated repression of NPY and AgRP mRNA levels in N-38 and N-42 neurons require either ER{alpha} and ERß or ER{alpha} alone, respectively, whereas the induction of NPY and AgRP in N-38 neurons is strictly ERß dependent, as assessed by ER-specific agonists and small interfering RNA knockdown of ER{alpha} or ERß. Through transient transfection analysis in N-38 neurons, we have mapped the estrogen-mediated repression of NPY to within –1078 of the 5' regulatory region of the NPY gene. Our results provide the first evidence that NPY and AgRP gene expression is directly regulated by estrogen in specific hypothalamic neurons, and that this regulation is dependent upon the ratio of ERß to ER{alpha}. The biphasic control of neuronal NPY/AgRP transcription may be a mechanism by which estrogen has distinct effects on both energy homeostasis and reproduction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NEUROPEPTIDE Y (NPY) IS an important regulator of energy homeostasis at the level of the hypothalamus (1, 2). A large body of research confirms the potent orexigenic effects of NPY on feeding behavior and the specific central effects and locations of NPY action (3). Any decrease in the levels of this peptide would thereby suppress appetite. Estrogen has long been shown to have a negative effect on feeding through actions in the hypothalamus (4); however, it is not clear which specific neuronal cell types expressing central neuropeptides are involved in this process. Estrogen potentially mediates its anorectic effects through a decrease in NPY release; however, a mechanism for this effect has not yet been described (5). It remains to be determined whether estrogen directly affects NPY neurons, and in which area of the brain. There is some evidence that NPY-expressing neurons in the hypothalamus concentrate 17ß-estradiol (E2) (6, 7) and that some NPY-positive cells in the hypothalamus also express ERs (8); therefore, distinct NPY neurons in the hypothalamus may be estrogen-responsive target cells. On the other hand, AgRP has been shown to be coexpressed with NPY in the hypothalamus and is also linked to the regulation of the melanocortin system through its potent orexigenic effects by antagonizing signaling at melanocortin 3 and 4 receptors in the hypothalamus (9, 10). However, estrogen regulation of agouti-related peptide (AgRP) synthesis has never been studied.

The GnRH neuron is a well-established target of estrogen, where it exerts both positive and negative feedback effects on GnRH secretion, as well as the pituitary release of LH and FSH (11). However, the mechanism behind the positive regulation of GnRH before the preovulatory surge is still under debate. It is generally assumed that afferent neuronal systems play a dominant role in the switch to positive regulation of GnRH (12). One of these cell types may indeed be the neuropeptide Y (NPY) neuron because NPY gene expression and protein accumulation increase immediately before the preovulatory GnRH surge (13, 14). Evidence also indicates that the sex steroid estrogen may play a role in the regulation of NPY synthesis (15, 16). However, the mechanisms by which estrogen exerts its effects on NPY neurons to positively regulate GnRH neurons remains to be elucidated. Furthermore, the potential role of AgRP in the regulation of reproductive function has never been investigated.

The actions of estrogens are primarily mediated through two specific nuclear receptor isoforms, ER{alpha} and ERß. The ERs act as homo- or heterodimers to modulate the transcriptional activity of target genes through their binding to estrogen response elements (17). Like other members of the ligand-activated nuclear receptor superfamily, the ERs have modular structures consisting of distinct functional domains (18). The two ERs share 58% homology in their LBD, which explains their similarities in estrogen binding; however, the N terminus shows little homology, 18% (19). This difference may suggest a possible divergence of transactivation between the ERs. In fact, the ERs have different transcriptional activities depending upon the ligand, cell type, or promoter context (17). The biological roles of ER heterodimers are not yet understood; however, it is known that ERß is a transdominant regulator of ER{alpha}-mediated gene expression (20, 21) and that if both ER isoforms are expressed the heterodimers predominate (22). This begs the question: does changing the ratio of ER isoforms in any particular cell type have an effect on gene expression?

To examine the mechanisms by which estrogen regulates NPY, and possibly AgRP synthesis, as well as its ability to play a role in the positive regulation of GnRH necessary for the preovulatory surge, one must be able to dissect the components of estrogen action within the individual cell types. In an attempt to produce a suitable model of the NPY/AgRP neuron, our laboratory has recently generated hypothalamic neuronal cell models, using retroviral transfer of simian virus 40 T-antigen into primary hypothalamic cell culture (23). Subcloning of the immortalized cells resulted in clonal populations of NPY/AgRP-expressing neurons. Two of these cell lines, N-38 and N-42, express neuronal cell markers, exhibit neurosecretory properties, and have been found to express and secrete NPY (23). In the present study, we investigated the regulation of the ERs, NPY, and AgRP by estrogen in these immortalized hypothalamic cell lines. Our results indicate that, in the N-38 neuronal cell line, estrogen induces a differential regulation of ER{alpha} and ERß transcription over 24 h, but only a repressive effect is seen in N-42 neurons. We have also demonstrated that both NPY and AgRP gene expression is directly regulated by estrogen in a biphasic manner in the N-38 neurons, corresponding to changes in the ratio of ER{alpha} to ERß. In addition, using small interfering (si) RNA against ER{alpha} and ERß, as well as ER-selective agonists, we show that the estrogen-mediated repression and induction of NPY and AgRP mRNA is an ER-dependent mechanism in N-38 and N-42 neurons. The estrogen-mediated down-regulation of NPY transcription occurs at the transcriptional level in N-38 neurons. These results present a novel mechanism by which estrogen may exert its distinct effects on feeding behavior and GnRH neuronal control in specific hypothalamic cell types.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of ERs and Other Hypothalamic Markers in N-38 and N-42 Neurons
We confirmed the presence of ER{alpha} and ERß mRNA and proteins in both the N-38 and N-42 neurons, using RT-PCR and Western blot analysis with specific antibodies to ER{alpha} and ERß (Fig. 1Go, A and B). HeLa cells, reported to be ER-negative, ER-positive mouse ovarian tissue and GT1–7 cells, served as controls. These results suggest that the N-38 and N-42 cell lines should be responsive to estradiol and therefore are appropriate models to study estrogen-mediated regulation of gene expression. We also analyzed the expression of an extensive list of neuropeptides, neuropeptide receptors, and enzymes associated with the synthesis of neurotransmitters in the N-38 and N-42 cell lines (Fig. 1CGo). We find expression of NPY and AgRP predominantly in these cell lines. However, there is evidence for vasointestinal peptide and melanocortin-concentrating hormone (MCH) expression in both cell lines, but N-42 cells also express galanin, urocortin, and somatostatin, indicating a unique phenotypic profile. Neither lines express proopiomelanocortin, cocaine- and amphetamine-regulated transcript, proglucagon, prothyroid-releasing hormone, arginine vasopressin, orexin, or pituitary adenylate cyclase-activating polypeptide. For the most part, both cells express similar receptors, including insulin, IGF-I, leptin, MCH-R1, NPY, and serotonin 5-HT2a receptors. Both lines exhibit evidence for {gamma}-aminobutyric acid synthesis, through glutamate decarboxylase expression, whereas only the N-42 cells express tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of catecholamines dopamine and norepinephrine, and dopamine transporter.


Figure 1
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  		Fig. 1. Identification of mRNA Transcripts and Protein for ER{alpha} and ERß and Other Hypothalamic Markers in Hypothalamic Neuronal Cell Lines

A, Total RNA was extracted and used as a template for RT-PCR with primers specifically designed to amplify ER{alpha} (344 bp) and ERß (243 bp). NTC, No template control. B, Cell lysates from N-38, or N-42 neurons were isolated and subjected to SDS-PAGE. Western blot analysis was performed with enhanced chemiluminescence using specific antibodies for ER{alpha} (67 kDa) and ERß (55 kDa). Immunoreactive bands were visualized using enhanced chemiluminescence. ß-Actin was used as a protein loading control. Protein from mouse ovary and GT1–7 neurons was used as a positive control. HeLa cells were used as a negative control. C, Phenotypic profile of mRNA expression for a number of neuropeptides, receptors, and enzymes, was generated in the N-38 and N-42 neurons. T-Ag, T-antigen; NSE, neuron-specific enolase; POMC, proopiomelanocortin; CART, cocaine- and amphetamine-regulated transcript; DAT, dopamine transporter; Gal, galanin; proGlu, proglucagon; proTRH, prothyroid-releasing hormone; AVP, arginine vasopressin; VIP, vasoactive intestinal peptide; Ucn, urocortin; SOM, somatostatin; PACAP, pituitary adenylate cyclase-activating polypeptide; TH, tyrosine hydroxylase; GAD, glutamate decarboxylase; MC4R, melanocortin 4 receptor; IR, insulin receptor; IGF1R, IGF-I receptor; ObR, leptin receptor b; NPY-R, NPY receptor; 5-HT2a, serotonin receptor; NTR1, neurotensin receptor 1. All data were generated by RT-PCR, with RT- and hypothalamic tissue as controls.

 
Regulation of ER mRNA Expression by Estrogen in N-38 and N-42 NPY Neurons, and GT1–7 GnRH Neurons
Little is currently known about the regulation of ER subtypes by estrogen itself. We first looked at the direct effect of estrogen on ER gene expression in novel NPY/AgRP-expressing cell models, N-38 and N-42. The N-38 and N-42 neurons were exposed to 10 nM E2 over a 24-h time course. In the N-38 neurons, an initial down-regulation of ERß mRNA expression from 1–6 h was observed, followed by a steady increase in ERß expression that peaked at 12 h (Fig. 2AGo). ER{alpha} mRNA levels, on the other hand, were significantly up-regulated at 2–4 h, and then subsequently repressed to 24 h. ER{alpha} and ERß protein levels were also regulated by estrogen in a similar manner (Fig. 2BGo). In the N-42 neurons, there was a steady repression of both ER{alpha} and ERß mRNA and protein (Fig. 2Go, C and D). When comparing the two cell lines, N-38 and N-42, it becomes evident that the ERs are differentially regulated by estrogen. Although they are both NPY-expressing cell lines, the regulation of ER mRNA is unique to each cell, indicating an inherent difference in phenotype between the cell lines. Interestingly, in GT1–7 cells, an established model of a GnRH neuron, we find that the ER subtype regulation follows a similar pattern to that of the N-38 NPY cell line (Fig. 2EGo).


Figure 2
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  		Fig. 2. Temporal Regulation of ER mRNAs and Protein by Estrogen in NPY-Expressing N-38 and N-42 Neurons, and GnRH GT1–7 Neurons

N-38 (A and B), N-42 (C and D), and GT1–7 (E) neurons were serum starved for 12–16 h before treatment with either 10 nM E2 or vehicle alone over a 24-h time course. At the indicated time points, total RNA or protein was extracted and used as a template for RT-PCR with primers specifically designed to amplify ER{alpha} and ERß or Western blot analysis, respectively. RT-PCR products were sized fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining. ER{alpha} and ERß mRNA levels were quantified by scanning densitometry and normalized to loading control (actin). Relative protein levels were also analyzed in the N-38 and N-42 neurons over 24 h. Specific antibodies toward to ER subtypes were used in Western blot analysis. All results shown are relative to corresponding control mRNA or protein levels (set to 1.0) at each time point and are expressed as mean ± SEM (n = 3 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.005. The ratio of ERß to ER{alpha} at each time point is indicated under the histograms.

 
Regulation of NPY and AgRP mRNA Expression by Estrogen in N-38 and N-42 Neurons
There is indirect evidence that estrogen is able to regulate NPY expression (15, 16); however, it is not yet known whether this regulation occurs directly at the level of the NPY neuron. We have therefore used our NPY-expressing N-38 and N-42 models to investigate whether estrogen can directly affect NPY transcription. N-38 and N-42 neurons were exposed to 10 nM E2 over 72 h. Analysis of the results in N-38 neurons indicates that NPY mRNA is biphasically regulated by estrogen (Fig. 3AGo). There is an initial down-regulation of NPY mRNA from 4–10 h, followed by a steady induction that peaks at 48 h, after which NPY mRNA levels begin to return to basal levels. This biphasic pattern of gene regulation appears to follow the estrogen-mediated regulatory pattern of the ERs; therefore, it can be postulated that changing the ER{alpha} to ERß ratio can cause a change in NPY transcription at the level of the NPY gene. On the other hand, NPY gene expression in the N-42 neurons is steadily repressed over the entire time course, with no appreciable change in this level of repression over 72 h (Fig. 3BGo). This highlights the probable importance of the increase in ERß to the up-regulation of NPY gene expression by estrogen, as seen in the N-38 neurons.


Figure 3
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  		Fig. 3. Estrogen Regulates NPY and AgRP mRNA in a Biphasic Manner in NPY-Expressing N-38 Neurons, but Only Represses NPY and AgRP mRNA in N-42 Neurons

N-38 (A and C) and N-42 (B and D) neurons were serum starved for 12–16 h before treatment with either 10 nM E2 or vehicle alone over a 72-h time course. At the indicated time points, total RNA was extracted and used as a template for real-time RT-PCR with primers specifically designed to amplify NPY or AgRP. Real-time RT-PCR products were amplified on an ABI Prism 7000. NPY and AgRP mRNA levels were quantified using the {Delta}{Delta}CT method and normalized to the internal control (actin). All results shown are relative to corresponding control mRNA levels (set to 1.0) at each time point and are expressed as mean ± SEM (n = 3 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.005.

 
We also studied the regulation of AgRP mRNA levels by estrogen in both cell lines. Although there is evidence that NPY and AgRP are coexpressed in the same neurons, little is known about the hormonal regulation of AgRP itself and whether it follows similar patterns to NPY. As above, N-38 and N-42 neurons were exposed to 10 nM E2 over 72 h. We find that AgRP mRNA levels are also regulated in a bimodal manner by estrogen in the N-38 cells, and NPY and AgRP gene expression patterns follow a similar timeline (Fig. 3CGo). In the N-42 cell line, like NPY, AgRP is repressed by estrogen over 72 h (Fig. 3DGo).

To study NPY gene regulation by estrogen at the transcriptional level, we used a region of the 5' flanking region of the mouse NPY gene cloned into a luciferase reporter gene, pGL2-enh, previously generated in our laboratory (24). The region cloned encompassed –1078 to +38 of the human NPY gene. To assess the cis-regulatory sequences necessary for the regulated expression of the NPY gene in the N-38 neurons, we transiently transfected pGL2-enh –1078 and pGL2-enh alone into the N-38 cell line and analyzed reporter gene activity. Luciferase activity of each construct was compared with that produced by the pGL2-enh alone, the parent vector, which displays negligible promoter activity. Reporter gene activity with the full-length promoter region from –1078 to +38 bp was approximately 8-fold greater than that of the pGL2-enh parent vector. A significant decrease in reporter gene activity occurs with 10 nM E2 treatment over 24 h (Fig. 4Go), indicating that the estrogen-mediated repression of the NPY gene may involve binding of transcription factors at the level of the NPY 5' regulatory region. When the treatment time was increased to 72 h in an attempt to detect the inductive effect of estrogen on NPY gene expression seen in the RT-PCR experiments, we did not observe any change in reporter gene activity with estrogen treatment (Fig. 4Go). We speculate that the mechanisms by which estrogen controls the NPY gene may differ between the early repressive effects vs. the longer-term up-regulation of transcription.


Figure 4
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  		Fig. 4. Estrogen-Mediated Repression of NPY Transcription Can Be Mapped to the Full-Length Human NPY 5' Regulatory Region in N-38 Neurons

The construct containing the full-length human NPY promoter (–1078 to +38) cloned into the promoterless pGL2-enhancer luciferase vector was transiently transfected into N-38 cells. After transfection, cells were treated with 10 nM estradiol or vehicle alone for 24 or 72 h. Luciferase activity was normalized to total cellular protein. Each value is expressed as fold-change relative to pGL2-enh vector alone and the vehicle-treated pGL2-enh –1078 NPY construct at the specific time point. Values are mean ± SEM from three experiments, each done in triplicate. **, P < 0.01 vs. untreated.

 
Estrogen-Mediated Regulation of NPY and AgRP Gene Expression Is Dependent on ER{alpha} and ERß in N-38 and N-42 Neurons
To deduce whether the ERs play a role in the regulation of NPY and AgRP gene expression, siRNA directed against ER{alpha} and ERß was used to selectively deplete the ERs and determine the effect on NPY and AgRP mRNA levels. siRNA was used at two specific time points that corresponded to the maximal level of repression and induction of neuropeptide mRNA expression (6 and 48 h for NPY, and 8 and 48 h for AgRP, respectively). At the 6-h time point, protein knockdown of both ER{alpha} and ERß blocked the estrogen-mediated repression of NPY mRNA, suggesting that both receptors are necessary for the early events (Fig. 5AGo). At the 48-h time point, protein knockdown of ERß (Fig. 5BGo) significantly attenuated the estrogen-mediated induction of NPY mRNA. In contrast, knockdown of ER{alpha} had no significant effect on NPY gene expression (Fig. 5BGo), as determined by RT-PCR. Only knockdown of ER{alpha}, but not ERß, at 8 h, blocked the repression of AgRP in N-38 cells (Fig. 5CGo), whereas only knockdown of ERß, but not ER{alpha}, had an effect on the up-regulation of AgRP mRNA levels at 48 h in the N-38 cell line (Fig. 5DGo). Using this same strategy, we found that the repression of NPY and AgRP by estrogen in the N-42 cell line could be linked to either ER{alpha} and ERß or ER{alpha} alone, respectively, at all time points studied (Fig. 6Go, A–D). The levels of ER subtype knockdown by siRNA in all of the experiments were similar and ranged from 75–90% (82% ± 4.2%) in the N-38 neurons, and 65–80% (72% ± 5.0%) in N-42 neurons, as determined by Western blot analysis using ER subtype-specific antibodies (Figs. 5EGo and 6EGo).


Figure 5
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  		Fig. 5. NPY and AgRP Gene Expression Is Dependent on ER{alpha} and/or ERß in N-38 Neurons

After transfection of siRNA directed toward ER{alpha} and ERß for 36 h for optimal knockdown of the ER subtypes, N-38 neurons were treated with either 10 nM E2 or vehicle alone for 6, 8, or 48 h. RNA and protein were isolated from the cells. RT-PCR analysis determined that knockdown of both ER{alpha} and ERß significantly blocked the estrogen-mediated repression of (A) NPY at 6 h, whereas only ER{alpha} is linked to repression of (C) AgRP mRNA at 8 h. RT-PCR analysis determined that knockdown of ERß significantly attenuated the estrogen-mediated increase of (B) NPY and (D) AgRP mRNA, whereas knockdown of ER{alpha} had no significant effect. All results shown are relative to control mRNA levels (set to 1.0) at each time point and are expressed as mean ± SEM (n = 3 independent experiments). *, P < 0.05. E, Cell lysates from N-38 neurons were isolated and subjected to SDS-PAGE. Western blot analysis demonstrated that the siRNA knocked down ER{alpha} by 79% (SE = 4.6%) in N-38 and 70% (SE = 4.1%) in N-42; and ERß by 84% (SE = 3.8%) in N-38 and 74% (SE = 5.8%) in N-42. Two representative Western blots are shown. Nonsense (N.S.) siRNA was used as a negative control.

 

Figure 6
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  		Fig. 6. NPY and AgRP Gene Repression Is Dependent on ER{alpha}/ERß or ER{alpha} in N-42 Neurons

After transfection of siRNA directed toward ER{alpha} and ERß for 36 h for optimal knockdown of the ER subtypes, N-42 neurons were treated with either 10 nM E2 or vehicle alone for 1 or 48 h. RNA and protein were isolated from the cells. RT-PCR analysis determined that knockdown of both ER{alpha} and ERß significantly blocked the estrogen-mediated repression of (A) NPY mRNA at 1 h, whereas knockdown of ERs at (B) 48 h had no significant effect on NPY gene expression. At 1 h, siRNA knockdown of either ER subtype had no effect on the levels of (C) AgRP mRNA. RT-PCR analysis determined that knockdown of ER{alpha} significantly attenuated the estrogen-mediated decrease of (D) AgRP mRNA at 48 h, whereas knockdown of ERß had no significant effect. All results shown are relative to control mRNA levels (set to 1.0) at each time point and are expressed as mean ± SEM (n = 3 independent experiments). *, P < 0.05. E and F, Cell lysates from N-42 neurons were isolated and subjected to SDS-PAGE. Western blot analysis demonstrated that the siRNA knocked down ER{alpha} by 79% (SE = 4.6%) in N-38 and 70% (SE = 4.1%) in N-42; and ERß by 84% (SE = 3.8%) in N-38 and 74% (SE = 5.8%) in N-42. Two representative Western blots are shown. Nonsense (N.S.) siRNA was used as a negative control.

 
ER-selective agonists were also used to further elucidate the role of the individual ERs in the estrogen-mediated regulation of the NPY gene at 6 and 48 h. Similar to the siRNA protocol, N-38 neurons were treated with 10 nM of the ER{alpha}-selective agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) and 10 nM of the ERß-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) for 6 and 48 h. At 6 h exposure, PPT caused a significant repression of NPY mRNA when compared with controls (Fig. 7AGo). This effect mimicked what was observed with estrogen treatment alone. DPN also slightly repressed NPY mRNA at 6 h; however, it did not reach statistical significance. At 48 h exposure, DPN significantly induced NPY mRNA expression, whereas PPT did not, as determined by real-time RT-PCR (Fig. 7BGo). These data suggest that both ER{alpha} and ERß are required for the estrogen-mediated repression of NPY, whereas the induction of NPY is dependent on ERß gene expression.


Figure 7
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  		Fig. 7. ER-Selective Agonists, PPT and DPN, Have a Significant Effect on NPY Gene Expression in N-38 Neurons

A, RT-PCR analysis demonstrated that 6 h treatment with 10 nM estradiol and 10 nM ER{alpha}-selective agonist, PPT, significantly repressed NPY mRNA expression in N-38 neurons. B, On the other hand, 48 h treatment with 10 nM estradiol and 10 nM of the ERß-selective agonist, DPN, significantly induced NPY mRNA expression. All results shown are relative to control mRNA levels (set to 1.0) at each time point and are expressed as mean ± SEM (n = 3 independent experiments). *, P < 0.05; **, P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Estrogen acts as a homeostatic feedback molecule between the gonads and the brain; however, the mechanisms by which this feedback occurs are unresolved. The GnRH neuron likely responds to estrogen directly, given that ERs are expressed in these cells in vivo (25, 26, 27), and the GnRH gene promoter can respond to estrogen in heterologous cell lines (28, 29). Furthermore, we have found that estrogen represses GnRH gene expression in a model of the GnRH neuron, the GT1–7 cell line (30), thereby indicating a direct negative feedback role at the level of the GnRH neuron itself. Studies in female ER knockout mice, with targeted disruption of either ER{alpha} or ERß, have demonstrated the necessity of ER{alpha} for the estrogen-mediated negative feedback of GnRH gene expression (31). However, estrogen also exhibits a positive feedback upon GnRH neurons to generate the preovulatory LH surge (32). Although estrogen is important for triggering ovulation, a coordinated release of modulatory factors is also necessary. Evidence for the necessity of NPY for the estrogen-mediated preovulatory surge has been provided through injection of antisense oligonucleotides directed against NPY mRNA into the arcuate nucleus of mice or primates (14, 33). Blockage of de novo NPY synthesis prevents the steroid-induced preovulatory rise in GnRH, and the surge release of LH that causes ovulation (34). In addition, basal LH secretion and all parameters of pulsatility have all been shown to be regulated by NPY through the use of antisense oligonucleotide technology (34). Studies of the NPY knockout mouse have also solidified the importance of the NPY neuron in facilitating ovulation, as in these mice the LH surge is attenuated (35). NPY neurons have also been shown to concentrate estradiol and express ERs in vivo (6, 7, 8), and in vitro (23); thus, the feedback effects of estrogen on GnRH neurons may be mediated via the NPY neuronal framework.

The positive actions of estrogen on the GnRH neuron are potentially indirect, through NPY neurons via the modulated expression of the ERs, ER{alpha}, and ERß. Although ER{alpha} and ERß share some functional characteristics, the molecular mechanisms regulating their transcriptional activity are distinct (36, 37, 38). This difference may be due to the fact that the two receptors share very little sequence identity between their amino-terminal transactivation domains (39). The ERs, acting as homo- or heterodimers, may activate target genes differentially depending on the ligand (38, 40, 41). Therefore, varying ratios of ER homo- and heterodimers can result in the tissue- and cell type-specific effects of estrogens, through the alteration of distinct gene expression patterns (42). In the present study, we demonstrate that ER{alpha} and ERß transcription is differentially regulated by estrogen in N-38 immortalized hypothalamic neurons. This finding allows us to speculate that alteration of the content, and thereby the ratio, of ER{alpha} and ERß, will potentially induce a differential effect on NPY gene expression. The concept of ER regulation by estrogen has already been shown in several tissues such as the uterus (43), liver (44), and aortic smooth muscle cells (45). Most relevant to this study is the regulation of ER gene expression in pituitary cells. Shupnik and colleagues have shown that the ratio of ER{alpha} and ERß, and a pituitary-specific truncated form of ER{alpha}, TERP-1, can change gene expression profiles in pituitary cells, which is postulated to be related to estrogen-positive feedback at this level of the HPG axis (46, 47, 48). These data support the idea that estrogen can act at the level of the pituitary, both positively and negatively, to influence the level of gonadotropin release in a cell type-specific manner.

A possible explanation for the differential regulation of ER gene expression in the hypothalamic cell lines is that ERß functions as a transdominant inhibitor of ER{alpha} transcriptional activity, as was observed by Hall and McDonnell (49). Interestingly, we also find that ERs are regulated similarly in the GT1–7 cells. This suggests that there may be some synchrony between the regulation of NPY and GnRH neurons by estrogen, similar to the temporal correlation seen during the pulsatile release of peptides from these neuronal cell types (50). We speculate that the change in ER{alpha} to ERß ratio can indeed have a direct influence over the longer-term transcriptional regulation of the NPY gene. Whether this effect is under transcriptional control, at the level of the NPY 5' regulatory region, or through a posttranslational mechanism, such as an estrogen-mediated change in NPY mRNA stability (51), is currently being investigated in our laboratory.

Although we have thus far focused on the potential regulation of the GnRH neuron by NPY, NPY is also requisite for the control of energy homeostasis (3). NPY is a potent orexigen because central administration of NPY stimulates feeding and repeated doses results in an increase in body weight (1, 2). However, the hormones and neuromodulators that directly affect NPY gene expression are not yet known. In addition to regulating reproductive function, estrogen has also been implicated in the regulation of components in adipose tissue, central control of feeding and energy homeostasis (52). Estrogen withdrawal through ovariectomy in rodents causes a temporary increase in food consumption, resulting in an increase in body weight (53). In humans, increased weight is often observed after menopause; however, postmenopausal women receiving estrogen replacement therapy have a decreased risk for insulin resistance, a reverse in the increased fat distribution, and the concomitant weight gain (54). Mice that lack ER{alpha} exhibit decreased energy expenditure and increased body weight and insulin resistance (55, 56). Furthermore, ER{alpha} null mice have been used to prove the involvement of ER{alpha} in estrogenic control of feeding behavior and body weight of female mice (56). Estrogen has long been shown to have a negative effect on feeding through actions in the hypothalamus (4); however, it is not clear which specific neuronal cell types expressing central neuropeptides are involved in this process. We speculate that estrogen can achieve its anorexigenic effects through the negative regulation of orexigenic neuropeptides, such as NPY and/or AgRP.

NPY and AgRP are coexpressed in neurons from the hypothalamus (57). AgRP is an endogenous antagonist of MC4R receptor signaling, thereby promoting food intake (9, 10). However the exact physiological role of AgRP, and whether it has any other functional significance, is not yet known. Few studies have analyzed hormonal regulation of AgRP synthesis. Similar to NPY, AgRP mRNA levels are down-regulated by leptin (58). The AgRP knockout mouse demonstrates no obvious phenotype, suggesting that this neuropeptide is not essential for energy homeostasis (59). However, more sophisticated knockout technologies that have taken a genetic approach to ablate not the AgRP peptide itself, but instead the neurons that make AgRP, and thus NPY, produce mice with hypophagia and leanness (60, 61, 62, 63). This indicates that perhaps NPY, AgRP, {gamma}-aminobutyric acid (also coexpressed in these neurons), and other potential as yet undefined factors, may be necessary for the coordinated regulation of energy homeostasis (64). No studies have yet been performed to study the temporal hormonal regulation of both NPY and AgRP concurrently. We find that these two peptides appear to be coordinately regulated by estrogen; however, the molecular mechanisms invoked for this regulation may differ at the transcriptional level because we demonstrate that both ERs are necessary to repress NPY mRNA, but only ER{alpha} is required for AgRP gene repression. However, in the N-38 cell line, we find that the induction of both NPY and AgRP at 48 h is an ERß-mediated event, which can be linked to the increase in ERß mRNA and protein levels, thereby altering the ER{alpha}: ERß ratio in the cell.

To our knowledge, no studies have yet been performed looking at ERß in NPY, and thus AgRP, neurons in situ, although it has been reported that the colocalization of ER{alpha} in NPY neurons is limited (65). We suggest that more studies are required with NPY neurons in vivo to conclusively decide whether they express ERs because technology has advanced significantly since the original studies, as has been shown with the recent acceptance of ER expression in the GnRH neuron. Although there have been many advances in technology, temporal analysis of direct estrogen-mediated regulation of ER gene expression in GnRH or NPY neurons in situ is not yet technically feasible, and as such, our neuronal cell lines represent the only practical model for these studies. Interestingly, the two NPY-expressing cell lines, N-38 and N-42, exhibit a differing pattern of ERß gene regulation. This regulation suggests that NPY-expressing neurons may exist as a heterogeneous population in the hypothalamus, as has been suggested for both NPY and GnRH neurons, depending upon the specific region of the brain, developmental pattern, and their afferent connections. Because we immortalized neurons from the entire hypothalamus to generate the clonal cell lines, we can only speculate on the precise area of the hypothalamus from which each clonal cell line originated due to the lack of known regional hypothalamic markers. Because the two lines respond to hormones uniquely, and have a distinctive phenotypic profile, it seems likely that they are from distinct nuclei in the hypothalamus. Certainly there is a precedent to functional heterogeneity in NPY neurons, as it has previously been shown that there are differential effects of steroids on NPY neurons in distinct nuclei of the hypothalamus (66). Furthermore, Kalra and colleagues (3, 5) have postulated that the anorectic effects of estrogen may be mediated through NPY neurons in the paraventricular nucleus of the hypothalamus, although other areas of the hypothalamus, especially the arcuate nucleus, where the vast majority of NPY-synthesizing neurons are located, could not be ruled out from these studies.

The results herein present a potential mechanism by which estrogen can regulate both feeding behavior and the HPG axis. We have shown that estrogen differentially regulates the ERs in two NPY/AgRP cell lines, and that the level of ERß expression is correlated to the level of peptide mRNA. We suggest that estrogen normally down-regulates NPY, and probably AgRP, gene expression to achieve its anorectic effects in the hypothalamus. However, the estrogen-mediated increase of ERß in specific cell types of the hypothalamus, potentially afferent to the GnRH neurons, is a possible mechanism by which the increase of NPY before the LH surge is accomplished. By knocking down ERß, we have also demonstrated that the necessary increase in NPY, and potentially AgRP, is attenuated. Although we cannot yet be certain that these mechanisms are those used in the intact hypothalamus, our novel immortalized neuronal cell lines are prime models to better understand the complex mechanisms involved in the regulation of specific gene expression patterns of distinct cell types in the hypothalamus. The findings presented herein may therefore be of significance for understanding the overall effects of estrogen in the cell types involved in reproductive function and energy homeostasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Reagents
N-38, N-42, and GT1–7 neurons were cultured in monolayer in DMEM (Invitrogen Life Technologies, Burlington, Ontario, Canada) and supplemented with 5% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT), 4.5 mg/ml glucose and penicillin/streptomycin and maintained at 37 C in an atmosphere of 5% CO2 as described (23). N-42 cells were generated in the same manner as the N-38 cell line, but subcloned after the initial report of the cell lines (23). Information regarding the phenotypic profile of these lines can be found on the web site CellutionsBiosystems.com. During steroid treatment, cells were serum starved for 12–16 h in phenol red-free DMEM, and then treatments were performed in phenol red-free medium supplemented with charcoal-stripped serum (30). E2 was obtained from Sigma-Aldrich Canada, Ltd. (Oakville, Ontario, Canada). ER{alpha}-selective agonist PPT and ERß-selective agonist DPN were obtained from Tocris Bioscience (Ellisville, MO). Final concentration of estradiol and ER-selective agonists in the treatments was 10 nM, whereas control cells were treated with comparable concentration of vehicle (ethanol, 0.0001% or 14 nM).

SDS-PAGE and Western Blot Analysis
N-38 or N-42 cells (80–90% confluent) were washed with ice-cold PBS and lysed in high-salt buffer [0.4 M NaCl, 20 mM HEPES (pH 8.0), 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1 mM phenymethylsulfonyl fluoride, 0.1% Nonidet P-40, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail for 40 min on ice]. Lysates were cleared at 12,000 x g for 20 min at 4 C and then treated for 4 min at 96 C with SDS-PAGE sample buffer containing ß-mercaptoethanol. Protein concentration was determined by the bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL). Forty-five micrograms of lysate protein were resolved on SDS-PAGE gels and blotted onto Hybond-C nitrocellulose membranes (Amersham Pharmacia Biotech, Baie d’Urfe, Quebec, Canada). The resulting blot was blocked with 5% skim milk in PBS containing 0.2% Tween 20, and incubated with primary antibody overnight at 4 C. The primary ER antibodies were used at 1 µg/ml of either polyclonal rabbit antimouse ER{alpha} or antirat ERß antisera (ER{alpha}, Figs. 1Go and 2Go: MC-20 (directed toward the carboxy terminal of the mouse ER{alpha}), no. sc-542; Santa Cruz Biotechnology, Santa Cruz, CA; ERß, Figs. 1Go and 2Go: PA1–310, Affinity Bioreagents Inc., Golden, CO). Immunoreactive bands were visualized with horseradish peroxidase-labeled secondary goat antirabbit antisera at 1:5000 dilution and enhanced chemiluminescence (ECL Kit Amersham Life Science, Inc., Oakville, Ontario, Canada) as described by the manufacturer. We also used another set of ER antibodies for Fig. 5Go. These were mouse monoclonal antihuman antibodies for ER{alpha} (10 µg/ml; Dako North America, Inc., Carpinteria, CA) and ERß (10 µg/ml; GeneTex Inc., San Antonio, TX). In this case, the immunoreactive bands were visualized with horseradish peroxidase-labeled secondary sheep antimouse antisera used at a 1:5000 dilution.

RT-PCR
N-38, N-42, and GT1–7 neurons were incubated in the presence of E2 (10 nM) or vehicle (ethanol) over a 24 h (for ER analysis) or 72 h (for NPY analysis) time course. The QIAGEN One-step RT-PCR kit was used according to manufacturer’s directions for ER amplification (QIAGEN Inc., Mississauga, Ontario, Canada), whereas real-time RT-PCR was used for NPY mRNA analysis. Total cellular RNA was isolated from N-38 or N-42 neurons by the guanidinium thiocyanate phenol choloroform extraction method (67). For the semiquantitative RT-PCR, the primers selected were: ER{alpha}—sense, 5'-GAA TTC AAT TCT GAC AAT CGA CGC CAG; antisense, 5'-GAA TTC GTG CTT CAA CAT TCT CCC TCC (344 bp fragment); ERß—sense, 5'-GAA TTC TAG CCA CCC ACT GCC AAT CAT; antisense, 5'-GAA TTC CAC ACC TTT CTC TCC TGG ATA (243-bp fragment); and {gamma}-actin: sense—5'-GCT CCG GCA TGT GCA A; antisense, 5'-AGG ATC TTC ATG AGG TAG T. For all cell lines, 100 ng of RNA was amplified. In the N-42 neurons, 36 and 30 PCR cycles were used to amplify ER{alpha} and ERß, respectively; in the N-38 neurons, 39 and 29 PCR cycles were used to amplify ER{alpha} and ERß, respectively. The {gamma}-actin gene was used as a loading control, and amplified for 20 cycles in the N-38 and N-42 neurons. ER{alpha} and ERß PCR products were electrophoresed in 2% agarose gels, stained in ethidium bromide solution [10 mg/ml ethidium bromide in 500 ml 1x TAE (Tris-acetic acid-EDTA)] for 15 min and destained in distilled water for 10 min. Gels were visualized under UV light, and quantified by densitometry using Scion Image for Windows software (Scion Corp., Frederick, MD).

Analysis of NPY and AgRP gene expression was performed over a 72-h time course where N-38 and N-42 neurons were continuously exposed to 10 nM E2, or to 10 nM agonists, PPT and DPN, for 6 or 48 h, as indicated. Cells were then harvested at the indicated times. Real-time RT-PCR was performed with SYBR green PCR master mix (Applied Biosystems, Inc., Streetsville, Ontario, Canada), according to the manufacturer’s instructions, and run on the Applied Biosystems Prism 7000 real-time PCR machine. Approximately 50 ng of template was used for the PCR, and the primer sequences for the NPY and AgRP transcripts are as follows: NPY-248-SYBR sense, 5'-CAG AAA ACG CCC CCA GAA; NPY-324-SYBR antisense, 5'-AAA AGT CGG GAG AAC AAG TTT CAT T; AgRP SYBR sense, 5'CGG AGG TGC TAG ATC CAC AGA; AgRP SYBR antisense, 5'-AGG ACT CGT GCA GCC TTA CAC. Real-time RT-PCR values were calculated by the {Delta}{Delta}CT method and normalized to {gamma}-actin mRNA levels at the corresponding time points. {gamma}-Actin sequences: actin SYBR sense, 5'-CTT CCC CAC GCC ATC TTG; and actin SYBR antisense, 5'-CCC GTT CAG TCA GAT CTT CAT.

Transient Transfections and Small Interfering (si) RNA-Induced Degradation of ER{alpha} and ERß
The full-length mouse NPY 5' flanking gene plasmid was generated by PCR as previously described (24). Transient transfections were performed using LipofectAMINE2000 (Invitrogen, Carlsbad, CA) according to manufacture’s instructions. The cells were incubated with the full-length NPY 5' flanking region (–1078NPY) in the pGL2-enh plasmid, or the pGL2-enh plasmid alone. After this 6 h transfection, the cells were treated with 10 nM E2 or vehicle and incubated for a further 24 or 72 h before harvesting. Relative luciferase units were normalized by total cellular protein and protein concentrations were determined using the bicinchoninic acid protein assay reagent. Luciferase assays were performed as previously described (24).

The siRNA species were purchased from New England Biolabs (Ipswich, MA) and designed to specifically target either ER{alpha} or ERß. 15 nM of ER{alpha} or ERß siRNA species were transiently transfected into N-38 neurons using LipofectAMINE2000 according to manufacturer’s directions and incubated for 36 h. The level of maximal knockdown was determined over a 48-h time course to choose this specific incubation time. A 174-bp DNA template derived from the cloning vector Litmus 28i was used as a nonspecific control (nonsense mixture). The cells were then treated with either 10 nM E2 or vehicle for 1, 6, 8, or 48 h. At these time points, cells were harvested from individual 60-mm plates, and RNA was extracted for RT-PCR, as well as protein from separate coordinate plates, to be resolved by Western blot analysis with indicated antibodies, as described above.

Statistical Analysis
Data were analyzed using one-way ANOVA by GraphPad Prism (GraphPad Software Inc., San Diego, CA) and statistical significance was determined using Tukey’s multiple comparison tests or Student’s t test with P < 0.05. Each experiment was performed on at least three to six separate occasions and was therefore considered independent experiments.


   ACKNOWLEDGMENTS
 
We thank members of the Belsham lab for critical reading of the manuscript. We gratefully acknowledge the gift of the GT1–7 cells from Dr. Pamela Mellon (University of California San Diego).


   FOOTNOTES
 
This work was supported by the Canadian Institutes for Health Research (CIHR). D.D.B. was a CIHR Scholar and now holds a Canada Research Chair in Neuroendocrinology and is a Canada Foundation for Innovation Researcher.

Disclosure: the authors have nothing to declare.

First Published Online May 4, 2006

Abbreviations: AgRP, Agouti-related peptide; DPN, 2,3-bis(4-hydroxyphenyl)-propionitrile; E2, concentrate 17ß-estradiol; ER, estrogen receptor; MCH, and melanocortin-concentrating hormone; NPY, neuropeptide Y; PPT, 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol; siRNA, small interfering RNA.

Received for publication January 16, 2006. Accepted for publication April 25, 2006.


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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  ERβ
Ligands:   17β-Estradiol



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