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Molecular Endocrinology, doi:10.1210/me.2005-0252
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Molecular Endocrinology 20 (7): 1547-1561
Copyright © 2006 by The Endocrine Society

Estrogen-Related Receptors-Stimulated Monoamine Oxidase B Promoter Activity Is Down-Regulated by Estrogen Receptors

Zhiping Zhang, Kevin Chen, Jean C. Shih and Christina T. Teng

Laboratory of Reproductive and Developmental Toxicology (Z.Z., C.T.T.), National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; and Department of Molecular Pharmacology and Toxicology (K.C., J.C.S.), School of Pharmacy, University of Southern California, Los Angeles, California 90033

Address all correspondence and requests for reprints to: Christina T. Teng, Ph.D., Head, Gene Regulation Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences/National Institutes of Health, P.O. Box 12233, MD E2-01, Research Triangle Park, North Carolina 27709. E-mail: teng1{at}niehs.nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although there are studies published about the neuroprotective effect of estrogen, little is known about the mechanisms and cellular targets of the hormone. Recent reports demonstrate that estrogen down-regulates the expression of monoamine oxidase A and B (MAO-A and MAO-B) in the hypothalamus of the Macaques monkey, both of which are key isoenzymes in the neurotransmitter degradation pathway. Additionally, estrogen-related receptor {alpha} (ERR{alpha}) up-regulates MAO-B gene expression in breast cancer cells. ERR{alpha} recognizes a variety of estrogen response elements and shares many target genes and coactivators with estrogen receptor {alpha} (ER{alpha}). In this study, we investigate the interplay of ERs and ERRs in the regulation of MAO-B promoter activity. We demonstrate that ERR{alpha} and ERR{gamma} up-regulate MAO-B gene activity, whereas ER{alpha} and ERß decrease stimulation in both a ligand-dependent and -independent manner. Ectopically expressed ERR{alpha} and ERR{gamma} stimulate the expression of MAO-B mRNA and protein as well as increase the MAO-B enzymatic activity in ER-negative HeLa cells. The ability of ERRs to stimulate MAO-B promoter activity was reduced in ER-positive MCF-7 and T47D cells. Several AGGTCA motifs of the MAO-B promoter are responsible for up-regulation by ERRs. Interestingly, ER{alpha} or ERß alone have no effect on MAO-B promoter activity but can down-regulate the activation function of ERRs, whereas glucocorticoid receptor does not. By using chromatin immunoprecipitation assay, we demonstrate that ERs compete with ERRs for binding to the MAO-B promoter at selective AGGTCA motifs, thereby changing the chromatin status and cofactor recruitment to a repressed state. These studies provide new insight into the relationship between ER{alpha}, ERß, ERR{alpha}, and ERR{gamma} in modulation of MAO-B gene activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN ACTIONS ARE mediated by at least two genetically different estrogen receptors, ER{alpha} (NR3A1) and ERß (NR3A2), which share similar, although divergent, cellular expression profiles in target cell nuclei and are functionally distinct (1, 2). Estrogen-activated ERs can interact with target genes directly by binding to specific, high-affinity estrogen-response elements (EREs) within promoters (reviewed in Ref. 3) or indirectly through protein-protein interaction with transcription factors, such as activating protein 1 (4, 5), Sp1 (6) and various other transcription factors (reviewed in Ref. 7 ; see references therein). In addition, estrogen acting through ER{alpha}, ERß, or possibly through other subtypes, participates in extranuclear signaling events in tissues of the cardiovascular, digestive, and neural systems (7, 8, 9). Based on highly conserved sequences of DNA-binding domain (DBD) to ERs, a subfamily, estrogen-related receptors (ERR{alpha}, NR3B1; ERRß, NR3B2; ERR{gamma}, NR3B3) was found (10) and classified into group III of the nuclear receptor superfamily (11). Although no natural ligands have been identified for these receptors, several synthetic compounds, either activating or repressing ERRs’ activities (12, 13, 14), have been identified. Recently, crystal structure analysis of the ligand-binding domain (LBD) predicted that ERR{gamma} may exist in a constitutively active form (15), whereas analysis of ERR{alpha} complexed with a coactivator peptide from peroxisome proliferator-activated receptor coactivator-1{alpha} (PGC-1{alpha}) reveals a transcriptionally active conformation in the absence of a ligand (16).

Emerging biological functions of ERRs including participation in bone morphogenesis (17), modulation of estrogen signaling (18, 19, 20), and involvement in energy metabolism (21, 22, 23, 24). The relationship of ERR{alpha} and estrogen response has been studied and was shown to repress ER{alpha}-mediated transactivation functions (25, 26). However, depending on the ERE and the surrounding elements, ERR{alpha} could also enhance the estrogen responsiveness (18, 27). Recently, many genes up-regulated by ERRs were discovered (22, 24, 28, 29); however, the roles of ERs in the regulation of those genes are not yet known. MAO-A and MAO-B were identified as targets of ERR{alpha} in MDA-MB-231 breast cancer cells by microarray analyses. When ERR{alpha} is overexpressed, it strongly induces the monoamine oxidase (MAO)-A and MAO-B expression (13).

MAO is a ubiquitous enzyme that oxidizes dietary amines and xenobiotics to prevent chemical toxicity and produces hydrogen peroxide as a by product. Hydrogen peroxide is a source of oxidative stress. Therefore, a low level of MAO activity is maintained in most of the cells except in specific tissue and developmental stages where the enzyme activity is essential. Two MAO isoenzymes have been identified in mammals with different substrate and inhibitor specificity and expression profiles. The tissue- and cell-specific expression of these isozymes was under the control of the promoter at the transcriptional level (30). Experiments on MAO-A or MAO-B knockout (MAO-A KO or MAO-B KO) mice indicated that the absence of each isoenzyme results in a different biochemical and behavioral phenotype (31). MAO-A KO mice showed an increasingly aggressive behavior; therefore it was used as a model to study impulsive human behavior (32). Interestingly, MAO-A and MAO-B double KO showed chase/escape and anxiety-like behavior different from the single MAO-B or MAO-B KO mice, implicating the importance of monoamine levels in both biochemical and behavioral phenotypes and suggesting that MAO-B is more important than we previously thought (33, 34).

Estrogen was suggested to have a modulatory effect in Parkinson’s disease because the disease is more prevalent, and progression of the disease is more rapid in males than in females. Furthermore, the beneficial effects of estrogen replacement in postmenopausal women, with regard to well-being and mood, may, in part, involve estrogenic action on MAO activity (reviewed in Refs. 35, 36, 37 ; see references therein). In rats, estrogen exerts a tissue-specific differential regulation of MAO-A and MAO-B activity. High doses of estrogen significantly decrease MAO-B activity in liver, kidney, and uterus with no significant changes in heart, lung, and small intestine (38). In addition, estrogen decreased MAO-A expression in the dorsal raphe region and decreased expression of both MAO-A and MAO-B in the hypothalamus of the rhesus monkey (39, 40). These data suggest that the transcriptional regulation of MAO by estrogen may play a role in serotonin or catecholamine neurotransmission and hence, mood, affect, or cognition in humans.

Considering that estrogen potentially modulates MAO activity, as well as the functional kinship and transcriptional cross talk between ERs and ERRs, we investigated the effect of ERR{alpha} and ERR{gamma} on MAO-B expression and the relationship between ER{alpha} or ERß and ERR{alpha} or ERR{gamma} on MAO-B gene transcription. We have shown here that ERRs up-regulate human MAO-B transcriptional activity, whereas ERs decrease the ERR{alpha}- and ERR{gamma}-stimulated transcriptional activity. This study provides new insight into MAO-B gene expression and the relationship between ERs and ERRs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ERR{alpha} and ERR{gamma} Stimulate MAO-B Gene Expression
The pattern of MAO-A and MAO-B expression in various human tissues was examined by using specific probes to MAO-A and MAO-B mRNA (Fig. 1Go) in a Northern blot analysis. MAO-A mRNA was detected at 5- and 2-kb regions in all tissues examined (upper panel). MAO-B mRNA was found in the heart, kidney, colon, skeletal muscle, and liver but not in the spleen and pancreas at the 3-kb region (lower panel). Thus, the two forms of MAO are differentially expressed. It was interesting to find that the kidney and heart, which have abundant MAO-B mRNA, also contain high levels of endogenous ERR{alpha}, ERR{gamma}, and PGC-1{alpha} (41, 42, 43, 44, 45, 46).


Figure 1
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  		Fig. 1. Expression Patterns of MAO-A and MAO-B in Human Tissues

Distribution of MAOs mRNA was determined by Northern blot with FirstChoice Northern Blot I (Ambion) as described in Materials and Methods. The premade blot contains 2 µg of poly(A) RNA per lane and the Millennium Marker band size at 6, 4, and 2 kb is indicated on the blot. After hybridization, the blot was exposed to x-ray film for 3 h for MAO-A mRNA (top panel) and 16 h for MAO-B (bottom).

 
The effect of ERR{alpha}, ERR{gamma}, and PGC-1{alpha} on the MAO-B expression was studied in HeLa cells, which contain endogenous ERRs (47) and MAO-B (Fig. 2Go). We found that the transcriptional activity of the MAO-B gene was stimulated by an ectopic expression of ERR{alpha}, ERR{gamma}, and PGC-1{alpha}, and the stimulations by ERR{alpha} and ERR{gamma} were reflected in the increase of mRNA (Fig. 2AGo), protein (Fig. 2BGo), and enzymatic activity (Fig. 2CGo). ERR{gamma} produced the strongest effect with a 5.6-fold increase followed by a 4.4-fold and a 2.2-fold increase in MAO-B mRNA by PGC-1{alpha} and ERR{alpha}, respectively (Fig. 2AGo, right panel). The same trend of stimulation, although less dramatic, was also found at the protein level, where the MAO-B protein was increased 2.86-fold by ERR{gamma} and 1.54-fold by ERR{alpha} (Fig. 2BGo, right panel). Additionally, MAO-B enzymatic activity was also enhanced by the expression of ERRs (Fig. 2CGo). These results demonstrated that ERR{alpha} and ERR{gamma} up-regulate endogenous MAO-B expression at the transcriptional level. It is not clear why the expression of ERRs does not stimulate MAO-B protein level and enzymatic activity to the same level as the mRNA. It is possible that the kinetics of protein synthesis, stability of the protein, and the detection methods could contribute to the observed differences.


Figure 2
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  		Fig. 2. ERR{alpha} and ERR{gamma} Up-Regulate MAO-B Expression

A, Northern blotting. HeLa cells were transfected with 2 µg of empty vector pSG5 (control) or expression vectors of ERR{alpha}, ERR{gamma}, and PGC-1{alpha} in 100-mm dishes. The experiments were repeated three times and a representative gel was shown. Total RNA (10 µg) was analyzed as described in Materials and Methods. The intensity of the MAO-B bands from the autoradiogram (left) was scanned and normalized with ß-actin. Scanning was done in an Innotech ChemiImager 5500 with signal spot densitometry according to the user’s manual. The control value was set as 1 arbitrary unit and presented as a bar graph (right). B, Western blotting. HeLa cells were transfected with empty vector, ERR{alpha}, or ERR{gamma} as in panel A. Protein (40 µg) of the whole-cell lysate from each experimental group was used to detect the ERR{alpha}, ERR{gamma}, MAO-B, and ß-actin in Western blotting (left). The experiment was repeated three times, and a representative gel is shown. The intensity of the MAO-B bands was scanned and normalized with the ß-actin bands. The scan was conducted with the Innotech ChemilImager 5500 with signal spot densitometry. The control value was set as 1 arbitrary unit and presented as a bar graph (right). Antibodies used to detect the expression of ERR{alpha} and ERR{gamma} were P277 and PG676 IgG fraction, respectively. C, Enzymatic catalytic activity. Protein (100 µg) from the experiments described in panel B was incubated with 10 µM 14C-labeled phenylethylamine at 37 C for 20 min. The organic phase containing the reaction product was extracted, and its radioactivity was measured by liquid scintillation spectroscopy. The control group radioactivity was set as 100%. The data are presented as means ± SD from three experiments. Statistical analyses were performed with Sigma Stat 3.1 software program. a and b indicate significant differences from the control (vector transfected) at P < 0.05 and P < 0.02, respectively.

 
ERR{alpha} and ERR{gamma} Stimulate the MAO-B Promoter Activity
To examine whether ERR{alpha} or ERR{gamma} stimulates the MAO-B promoter through specific response elements, we searched a 2.1-kb region of the human MAO-B promoter and found two AGGTCA motifs at the distal region (–1468 and –1762) and an AGGTGACCT motif, consisting of an overlapping AGGTGA and a reverse oriented AGGTCA, at the proximal region (–289/–286) (Fig. 3AGo). These elements could be potential binding sites of ERR{alpha} and ERR{gamma} because the ERR response elements contain an AGGTCA or TGACCT core motif. To test this hypothesis, the wild-type (wt) construct containing the MAO-B promoter sequence between –2099 and –99 bp was used in most of the studies. In addition, deletion mutant constructs and single or multiple site-specific mutation constructs of the human MAO-B promoter-reporter were also used. As expected, ectopic expression of ERR{alpha} and ERR{gamma} increased the MAO-B promoter activity in a dose-dependent manner, and the activity was able to be stimulated to 14- and 51-fold above the control, respectively (Fig. 3BGo). As shown by Western blotting with the myc antibody, both ERR{alpha} and ERR{gamma} expression constructs were efficiently transcribed and translated in the HeLa cells (Fig. 3BGo, inset), suggesting that the differences in ERR{alpha} and ERR{gamma} stimulation were not due to the differential expression, but to the functional difference of the ERR{alpha} and ERR{gamma}.


Figure 3
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  		Fig. 3. ERR{alpha} and ERR{gamma} Stimulate MAO-B Promoter Activity

A, Diagrammatic representation of the MAO-B promoter. Four AGGTCA motifs are marked. The core promoter consists of a TATA box and two clusters of overlapping Sp1 sites separated by a CACCC element. Promoter-reporter wt and mutant constructs (D4, D5, and D10) were previously described (75 ). Mutations of GG or CC of the AGGTCA motifs were marked as m1, m2, m3, and m4 and were described in Materials and Methods. B, Ectopic expression of ERR{alpha} and ERR{gamma} stimulates the transcriptional activity of MAO-B promoter (wt). The vector (50 ng), ERR{alpha}, or ERR{gamma} expression plasmids (5, 20, 50, and 100 ng) with the MAO-B promoter reporters were transfected into HeLa cells for 24 h as described in Materials and Methods. The reporter activity was measured and normalized with the internal control. Data are presented as the mean ± SD from three independent experiments with duplicate samples. Inset, Myc-tagged ERR{alpha} or ERR{gamma} (50 ng of expression plasmids) was transfected into HeLa cells for 24 h and the whole-cell lysate was prepared. The ectopically expressed ERR{alpha} and ERR{gamma} were detected by myc antibody in Western blotting. C, Effect of 4-OHT on the transactivation function of ERR{alpha} and ERR{gamma}. HeLa cells were transfected with ERR expression plasmids (50 ng) and the MAO-B promoter reporters (100 ng). (Legend continues on next page.) After transfection, cells were treated with vehicle, 10–9, 10–8, 10–7, 10–6, and 10–5 M 4-OHT for 24 h, and the MAO-B promoter reporter activity was measured. Data are presented as means ± SD from three experiments with duplicate samples. D, AF2 domains of the ERR{alpha} and ERR{gamma} are important in their transactivation function. The wt or mutant ERR{alpha} and ERR{gamma} were tested with the MAO-B promoter reporter (wt). AF2m, Mutation at L413A and L418A of the ERR{alpha} AF2 domain; P-boxm, mutation at E97G/A98S/A101V within the predicted P-box of the ERR{alpha} DBD; AF2{Delta}, deletion of the last nine amino acids of the ERR{gamma}, which contains the AF2 domain. The ERR mutant constructs were expressed comparably in a transient transfection study (48 49 ).

 
To test whether blocking the ERRs’ transcriptional activity could impair its ability to transactivate the MAO-B promoter, we treated the cells with 4-hydroxytamoxifen (4-OHT), a known inverse agonist for ERR{gamma} for 24 h after transfection. The activity of ERR{gamma} was inhibited by increasing concentration of 4-OHT, as was its ability to stimulate the MAO-B promoter (Fig. 3CGo). The dose required to block the stimulation by ERR{gamma} is in the range with the dose needed to block its transactivation activity (12). Because 4-OHT has no inhibition function over ERR{alpha}, it did not interfere with its ability to transactivate the MAO-B promoter. These results reaffirmed that the MAO-B promoter is activated by ERRs.

The activation function 2 (AF2) domain of most nuclear receptors potentiates significant transcriptional activities. We tested the importance of the AF2 domain in ERR{alpha} or ERR{gamma} on the MAO-B promoter. It was surprising that the mutations of ERR{alpha} AF2 domain [L413A and L418A, (48)] have only a partial effect on its activation function, whereas a p box mutation causes severe loss of activation function (Fig. 3DGo, left panel). This suggests that the DNA binding and AF2 domains of the receptor are required during stimulation of the MAO-B promoter. In contrast, deletion of the AF2 domain from ERR{gamma} (deletion of the last nine amino acids of the ERR{gamma} AF2 domain) completely abolished its transactivation function (Fig. 3DGo, right panel), even though the receptor could still bind the DNA element (49). These studies showed the importance of AF2 in stimulation of MAO-B promoter by the ERR{alpha} and ERR{gamma}. {Delta}AF2 may act as dominant-negative receptor in the activation of MAO-B promoter transcription.

Next, we tested to determine which regions of the MAO-B promoter are responsible for the ERR{alpha} or ERR{gamma} function. We used the previously constructed deletion mutant D4, which contains the core promoter and the proximal AGGTGACCT, D5, which contains the core promoter only, D10, which has an internal deletion including the two distal AGGTCA motifs, and newly produced site-specific mutation mutant constructs (Fig. 3AGo). All the mutant constructs and wt have very low basal promoter activity (data not shown). However, the ability of ERR{alpha} or ERR{gamma} to stimulate the deletion mutant promoters was impaired (Fig. 4AGo, left and middle panels). This result showed that the AGGTCA motif-containing regions are important in ERR{alpha}- and ERR{gamma}-stimulated activities, and the presence of these motifs may be required for a full activation function. Assuming that the ectopic expression of either ERR{alpha} or ERR{gamma} increases wt reporter activity to 100%, ERR{alpha} or ERR{gamma} can only increase the D4 activity to 22% and 50%, whereas they can increase D10 to 38% and 47%, respectively. These results showed that the potential ERR{alpha}- and ERR{gamma}-binding regions of the MAO-B promoter are important in mediating ERR{alpha}- and ERR{gamma}-stimulated activity. Interestingly, in the presence of both ERR{alpha} and ERR{gamma}, deletion of the distal AGGTCA motifs reduced the promoter activity only 20–30% (compare D4 and D10 with wt in Fig. 4AGo, right panel). In the absence of all four AGGTCA motifs (D5), the ERR{alpha} or ERR{gamma} by itself did not activate the MAO-B promoter. However, together they were able to slightly stimulate the promoter activity. This deletion study could not exclude the presence of other unknown or unnoticeable response elements that are mediating the ERRs’ activities especially under the condition that both ERR{alpha} and ERR{gamma} are present. To evaluate the contribution of individual AGGTCA motifs in ERR-stimulated activities, we made single, double, or triple mutations of these motifs (Fig. 3AGo). Mutations at the CC dinucleotide of the TGACCT motif (m4) has a dramatic effect on the transactivation function of ERR{gamma}, whereas the other three sites, mutated either individually or together, have little or no effect (Fig. 4BGo, right panel). In contrast, mutation of these motifs have only moderate effect on ERR{alpha} function (Fig. 4BGo, left panel) suggesting that other sites may participate in the ERR{alpha} binding and activation of the MAO-B promoter or ERR{alpha} and ERR{gamma} may be acting through a different mechanism.


Figure 4
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  		Fig. 4. The AGGTCA Motifs of the MAO-B Promoter Are Important for ERR{alpha} and ERR{gamma} Response

A, Effect of AGGTCA deletion. The proximal and distal AGGTCAs of the MAO-B promoter were deleted as shown in Fig. 3AGo. HeLa cells were transfected with the MAO-B promoter reporters (wt or D4, D5, or D10) and ERR{alpha} or ERR{gamma} expression plasmids individually or in combination for 24 h. The reporter activities were measured and normalized with internal control. Data are presented as percentage of activation with the wt set as 100%. B, Effect of AGGTCA mutation. The MAO-B promoter containing single, double, or triple mutations at the AGGTCA motifs located at m1, m2, m3, and m4 sites (Fig. 3AGo) were transfected with either ERR{alpha} or ERR{gamma} expression constructs into HeLa cells, and the reporter activities were measured. Activity of the wt MAO-B promoter was set as 100%. The experiments were repeated three times with duplicate samples. Data are presented as means ± SD.

 
ERs Down-Regulate the Transactivation Function of ERR{alpha} and ERR{gamma} on the MAO-B Promoter
Based on the close structural and functional relationship of ERs and ERRs as well as the potential estrogen effect on the MAO-B activity, we investigated whether ERs have an effect on the activity of MAO-B promoter in the presence or absence of estrogen (Fig. 5Go). The transient transfection experiments showed that ERs do not stimulate, but repress, the MAO-B promoter activity in the presence of estrogen. On the contrary, ERRs are capable of stimulating the MAO-B promoter with or without the presence of estrogen or ER’s antagonist ICI182,780 (ICI) (Fig. 5AGo). In HeLa cells overexpressing ER{alpha}, 17ß-estradiol (E2) further repressed the MAO-B promoter activity, and in the cells overexpressing ERß, E2+ICI showed additional repression. To test for a potential modulatory role of ER on the ERR-regulated MAO-B promoter, we coexpressed ERs and ERRs. The human MAO-B promoter was cotransfected into HeLa cells with ERR{alpha} and ER{alpha} or ERß (Fig. 5BGo, left panel), or ERR{gamma} and ER{alpha} or ERß (Fig. 5BGo, right panel). After transfection, cells were either treated with vehicle, E2, or E2 plus ICI. In the absence of ERs and E2, the ERR-stimulated promoter activity was set as 100%. Ectopic expression of ER{alpha} brought down the ERR{alpha}-stimulated MAO-B promoter activity to half, and additional repression was seen when E2 was added (Fig. 5BGo, left panel). The ER{alpha}-E2-dependent inhibition could be reversed by ICI. Ectopic expression of ERß also decreased the ERR{alpha}-stimulated MAO-B promoter activity to 49%, and to 30% in the presence of E2. Unexpectedly, ICI did not reverse the E2-dependent repression but further reduced the promoter activity to 17% (Fig. 5BGo, left panel). These experiments were repeated with ERR{gamma} (Fig. 5BGo, right panel), and a similar pattern of repression by ER{alpha} was found (44% without E2, and 21% with E2). Again, ICI could block the repression caused by E2. ERß strongly inhibited the ERR{gamma}-stimulated MAO-B promoter activity (Fig. 5BGo, right panel) and ICI enhanced the ERß effect. These results demonstrated that the down-regulation of ERR{alpha} or ERR{gamma}-stimulated transcriptional activity of the MAO-B promoter by ER{alpha} and ERß is complicated and may involve several mechanisms.


Figure 5
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  		Fig. 5. ERs Down-Regulate the ERR-Stimulated MAO-B Promoter Activity

A, The activity of MAO-B promoter (wt) is stimulated by ERR{alpha} and ERR{gamma} but not by ER{alpha} and ERß with or without the presence of estrogen. The fold of activation was calculated with the activity of the empty vector set as 1 and the data are presented as the mean ± SD from three independent experiments with duplicate samples for each experiment. Statistical analyses were done with Student’s t test. a Indicates the significant difference from no E2; P < 0.05. B, ER{alpha} and ERß repress the transactivation function of ERR{alpha} and ERR{gamma} on the MAO-B promoter, and estrogen further enhances the repression. After transfection, cells were either treated with 10–8 M E2 alone or together with 10–6 M ICI for 24 h before assay. The RLU of the ERR{alpha} and ERR{gamma} was set as 100%, and the data are presented as the mean ± SD from three independent experiments with duplicate samples for each experiment. a Indicates the significant difference from no E2; P < 0.05. C. LBD of the ER{alpha} is needed for the ligand-dependent and -independent repression function. Upper panel, After transfection, cells were treated with vehicle or 10–8 M E2 for 24 h before assay. The RLU of ERR{alpha} or ERR{gamma} was set as 100%, and the data are presented as mean ± SD from three independent experiments with duplicate samples. Diagrammatic presentation of the ER{alpha} deletion mutants (HE11, HE15, and HE19) is in the lower panel.

 
To map the ER{alpha} domain that is responsible for the down-regulation, we used three different deletion mutant constructs (Fig. 5CGo, low panel). ER{alpha} lacking the DBD (HE11) or C-terminal region (HE15) were not able to inhibit the activity of ERR{alpha} (Fig. 5CGo, upper left panel) or ERR{gamma} (Fig. 5CGo, upper right panel); however, the deletion mutant containing the DBD and LBD regions (HE19) was able to repress ERR{alpha}- and ERR{gamma}-induced MAO-B promoter activity, implicating the importance of these two regions in the repression function. It was interesting to find that the deletion mutant, HE11, did not inhibit, but also stimulated, the activity of the ERRs, especially in the case of ERR{alpha}. It should be noted that the ER{alpha} mutant constructs used in this study were the original HEO version with G400V mutation (50). The HE19 inhibited the activation function of ERRs on the MAO-B promoter similar to the wt ER{alpha} (HEGO version). Although ERß was not mapped in this way, it may prove to be an interesting investigation in the future.

Endogenous ERs Repress ERRs Stimulated MAO-B Promoter Activity
Here, we explored whether endogenous ERs suppress the transactivation function of ERRs on the MAO-B promoter. We first examined the endogenous levels of ERs and ERRs in MCF-7, HeLa, and T47D (Fig. 6AGo). Western blotting showed that ER{alpha} and ERß are present in MCF-7 and T47D but not in HeLa cells, which is consistent with the literature. ERR{alpha} and ERR{gamma} were abundant in HeLa cells and easily detected by a specific antibody using the whole-cell lysate, whereas, conversely, in MCF-7 and T47D, ERR{alpha} and ERR{gamma} were barely detectable under the same conditions. In MCF-7 and T47D, ERRs showed a modest stimulation (3- to 5-fold) of MAO-B promoter activity in contrast to the robust stimulation of 10- to 20-fold observed in the HeLa cells (Fig. 6BGo). The reduced response, at least in part, could be due to the presence of endogenous ERs that inhibit the ERR function in an unliganded state. Addition of E2 to ER-positive MCF-7 cells further reduced the ability of ERRs to stimulate the MAO-B promoter activity, whereas ICI blocked the estrogenic effect (Fig. 6CGo). These observations are consistent with the notion that ERs are involved in down-regulation of ERRs’ transactivation function on the MAO-B promoter. Interestingly, expression of glucocorticoid receptor (GR) in HeLa cells has no repressing effect contrary to the ER expression. In fact, GR has a modest enhancing effect on the activity of an ERR{alpha}-stimulated activity, whereas in the presence of dexamethasone (DEX), a stronger enhancing effect on an ERR{gamma}-stimulated MAO-B promoter activity was found whether or not GR is overexpressed (Fig. 6DGo). To demonstrate that GR, either endogenous or ectopically expressed, is functioning in HeLa cells, we transfected the reporter constructs carrying the GR response element in the absence and in the presence of DEX. An expected stimulation of the reporter constructs was detected (data not shown). Ultimately, the repressive effect of ERs on the ERR-mediated stimulation of MAO-B promoter in ER-positive cells supports the transient transfection study in ER-negative cells, and the observed effect is promoter specific rather than the general "squelching" of limited coactivators.


Figure 6
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  		Fig. 6. Endogenous ERs Suppress the Transactivation Function of ERRs on the MAO-B Promoter

A, Endogenous levels of the ERs and ERRs in MCF-7, HeLa, and T47D. Protein (40 µg) from the whole-cell lysate of each cell line was Western blotted with specific antibodies. B, Differential response of MAO-B promoter to ERRs in ER-positive, MCF-7 and T47D, and ER-negative, HeLa, cells. Cells were transfected with MAO-B promoter reporter and either ERR{alpha} or ERR{gamma} as described in Materials and Methods. The reporter activity is presented as fold of activation against empty vector control. C, Estrogen enhances the ability of endogenous ERs to suppress the ERR-stimulated MAO-B promoter activity. MCF-7 cells were treated 24 h after transfection with 10–8 M E2 or E2 plus 10–6 M ICI, and the Luc activity was measured 24 h later. D, GR does not repress the transactivation function of ERRs on the MAO-B promoter. The GR expression constructs (50 ng) were cotransfected with the ERRs (50 ng) and the MAO-B-Luc reporter (100 ng) into HeLa for 4 h, and the cells were treated with DEX at 10–7 M for 24 h before the reporter activity was determined. Data are presented as mean ± SD from three experiments with duplicate samples.

 
ERs Induce Chromatin Changes of the MAO-B Promoter
The functional study has identified important regions of the MAO-B promoter that are needed in ERR stimulation. To investigate whether ERs and ERRs bind chromatin at these regions, we performed chromatin immunoprecipitation (ChIP) assay. Based on the location of the AGGTCA motifs, we prepared probes to examine four regions of the MAO-B promoter (Fig. 7AGo). ChIP1 covers the proximal AGGTGACCT, and ChIP2 and ChIP3 cover the two distal AGGTCA motifs, respectively. The DNA was sheared to a 200- to 1000-bp fragment in the ChIP assay, so the resolution between ChIP2 and ChIP3, which are 300 bp apart, may not be able to be clearly delineated. ChIP4 is located more than 7 kb upstream from the promoter region and served as a negative control.


Figure 7
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  		Fig. 7. Ligand-ER{alpha} and ERß Induce Chromatin Structure Change and Coregulator Recruitment of the MAO-B Promoter

A, MAO-B promoter regions examined by ChIP assay. Location of the proximal TAGGTGACCT (ChIP1), distal AGGTCA (ChIP2 and ChIP3), and unrelated (ChIP4) regions are marked. B, Detection of ERR{alpha} and ERR{gamma} and ER{alpha} and ERß on the MAO-B promoter at the AGGTCA-containing regions. Left panel, Binding of ectopically expressed Myc-ERR{alpha} and ERR{gamma} to the MAO-B promoter are detected by Myc antibody. Middle panel shows that the endogenous ERR{alpha} and ERR{gamma} are detected on the MAO-B promoter by specific anti-ERR{alpha} and anti-ERR{gamma} antibodies, respectively. Right panel shows that the ectopically expressed ER{alpha} and ERß are detected by specific anti-ER{alpha} and anti-ERß antibodies, respectively. The normal rabbit IgG serves as negative antibody control in all of the assays. C, ERs and ERRs compete for binding to selective AGGTCA-containing regions of the MAO-B promoter. HeLa cells were transfected with either ER{alpha} or ERß for 4 h and treated with 10–8 M E2 for 24 h. The occupancy of the endogenous ERRs on the MAO-B promoter was detected by specific anti-ERR{alpha} and anti-ERR{gamma} antibodies. D, Ligand-ER{alpha} represses histone acetylation and enhances corepressor recruitment of the MAO-B promoter. Ac-H3, Antiacetylated histone 3; Ac-H4, antiacetylated histone 4; mSin3A, anti-mSin3A; CBP, anti-CBP; SMRT, anti-SMRT; and RIP140, anti-RIP140. The number of PCR cycles is indicated. E, ERß represses histone acetylation and enhances corepressor recruitment of the MAO-B promoter. The antibodies used in the ChIP assays are identical to those in panel D. F, Effect of estrogen on occupancy of endogenous ERs and ERRs at the MAO-B promoter. MCF-7 cells were treated with 10–8 M E2 for 24 h before the ChIP assays were performed. Intensity of the band was scanned in an Innotech ChemiImager 5500 with signal spot densitometry according to the user’s manual. Untreated sample is set as 1.

 
First, we ectopically expressed the Myc-ERR{alpha} or ERR{gamma} in the HeLa cells and then performed ChIP assays with a monoclonal anti-Myc antibody. This approach allowed us to detect translated ERRs but not the endogenous ERRs. The Myc-tagged ERR{alpha} and ERR{gamma} were detected at ChIP1, ChIP2, and ChIP3, but not at ChIP4 (Fig. 7BGo, left panel). Endogenous ERRs were then tested. Binding of the endogenous ERRs at ChIP1, 2, and 3 were also demonstrated by using specific anti-ERR{alpha} or anti-ERR{gamma} antibodies (Fig. 7BGo, middle panel). These results demonstrated that ERR{alpha} and ERR{gamma} indeed interact with AGGTCA-containing regions of the MAO-B promoter at the native chromatin structure. Likewise, the ectopically expressed ERß was also bound to the ChIP1, 2, and 3 but not the ChIP4. Interestingly, ectopically expressed ER{alpha} was only detected at the ChIP1 (Fig. 7BGo, right panel). As negative control, normal rabbit IgG was unable to immunoprecipitate any DNA-protein complexes on the MAO-B chromatin. These results support the involvement of ERRs and ERs on the MAO-B promoter activity in vivo by binding to the same location of the MAO-B promoter. To address what happens to the ERR{alpha} or ERR{gamma} occupancy of these ChIP regions when ERs are ectopically expressed, we transfected the HeLa cells with either ER{alpha} or ERß expression constructs and treated them with 10–8 M E2 for 24 h before performing the ChIP assay (Fig. 7CGo). The results showed that ER{alpha} only displaces ERR{alpha} or ERR{gamma} from the proximal ChIP1 region, whereas ERß displaces the ERRs from both proximal and distal ChIP1–3 regions, which is in agreement with the detection of ERR and ER occupancy on the MAO-B promoter. These results suggest that a competition of these two classes of receptors occurs on the chromatin of ERR-binding sites.

Function of the ERs is mainly dependent on associated coregulators, some of which possess chromatin modification capability (reviewed in Ref. 51 ; see references therein). An important step in the modification of the chromatin is by histone acetylation. To examine the effect of liganded ER{alpha} or ERß on the histone acetylation and coactivator or corepressor recruitment to the MAO-B promoter, we used various antibodies related to the histone acetylation status (Ac-H3 and Ac-H4) and coregulator recruitment [CREB-binding protein (CBP), silencing mediator of retinoid and thyroid hormone receptor (SMRT), mSin3A and receptor interacting protein 140 (RIP140)] in the ChIP assays. HeLa cells that contain endogenous ERRs and a low level of MAO-B were transfected with ER{alpha} or ERß expression constructs and treated with E2 for 24 h before the assay. In the presence of E2-ER{alpha} (Fig. 7DGo) or E2-ERß (Fig. 7EGo), a decrease of acetylated histone 3 and 4 and an increase of mSin3A, an important component of the deacetylation complex, at the ChIP1, 2, and 3 were observed. The increase of mSin3A was clearly demonstrated with the 40-cycle PCR product. Consistent with the repressed chromatin status, coactivator CBP content was decreased, and corepressors such as SMRT and RIP140, increased at the MAO-B promoter even though the ChIP region of the coregulator exchange did not match exactly (Fig. 7Go, D and E, low panel, the 40-cycle PCR product). ChIP4, which is 7 kb upstream from the MAO-B promoter, has low levels of acetylated histone 4 and barely detectable acetylated histone 3 regardless the ectopic expression of ERs and the presence of E2. The coregulators were not detected at this region either. To study the occupancy of the MAO-B response elements by endogenous ERs and ERRs in the presence and absence of E2, we performed ChIP assay with MCF-7 cells (Fig. 7FGo). Like the HeLa cells, ERR{alpha} and ERR{gamma} were detected at ChIP1, ChIP2, and ChIP3 but not ChIP4 region. After E2 treatment, occupation of ERRs at these regions was reduced by at least 50%. Endogenous ER{alpha} was also detected at ChIP1 but not the other three regions, whereas ERß was detected at ChIP1–3, supporting the transfection experiments (Fig. 7BGo, right panel). As expected, E2 treatment enhanced the occupancy of ERs at the AGGTCA-containing regions 2- to 4-fold. These results agree with the observations in HeLa cells that endogenous ER{alpha} only binds to ChIP1, whereas ERß, ERR{alpha}, and ERR{gamma} bind ChIP1–3. Furthermore, estrogen enhances the binding of ERs and reduces that of ERRs. These ChIP assay data provide a mechanism to accompany the functional study and show that the liganded ERs down-regulate the MAO-B promoter activity via competition for binding with ERRs to the AGGTCA motifs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The ERRs have been shown to modulate ER-mediated estrogen action (reviewed in Ref. 20 ; see references therein). The present study demonstrated that the ER could decrease the transactivation activity of ERRs on the MAO-B promoter. We showed that ERRs stimulate the activity of the MAO-B promoter and that ERs block the stimulation in both a ligand-dependent and -independent manner. Three regions of the MAO-B promoter containing the AGGTCA motif are involved in ERR stimulation, and the proximal TGACCT motif is most important. We showed that the ERs compete with ERRs to bind the chromatin of those regions and, in the presence of ERs and E2, they assume a repressed chromatin status. These results are the first to show that ERs inhibit the stimulation of the MAO-B gene promoter by ERRs.

MAO-B breaks down amines derived from internal and exogenous sources. Although the role of MAO-B in peripheral tissues is not clearly defined (52), it may be important in controlling the local level of neurotransmitters, which, in turn, controls various physiological processes such as sodium reabsorption, blood pressure, and pregnancy. MAO-B is highly expressed in the central nervous system and has been linked to several neurodegenerative diseases and psychiatric disorders (53). Estrogen was suggested to play a protective role by regulation of MAO expression (38, 39). In addition, a negative correlation between serum estrogen levels and the MAO activities of platelets was found in humans (54) despite the lack of molecular evidence that estrogen can regulate the MAO-B expression. Response elements of the MAO-B promoter have been partially characterized, and two clusters of overlapping Sp1 sites near the initiation start site were shown to be necessary for the regulation of MAO-B expression (55). These sites could mediate the estrogen action through protein-protein interaction between Sp1 and ER (6). Furthermore, variations of AGGTCA motifs of half-ERE sites were found within 2 kb of the promoter region, which could also be target sites of estrogen action. Surprisingly, when the MAO-B promoter containing these elements was tested in HeLa cells in transient transfections, the promoter did not respond to estrogen stimulation with or without ectopically expressed ERs, but was stimulated by the expression of ERRs (Fig. 5AGo). In the presence of ERR{gamma} inverse agonist, 4-OHT, ERR{gamma} but not ERR{alpha} lost its activation effect on the MAO-B promoter (Fig. 3CGo). This observation strongly corroborated the transactivation function of ERRs on the MAO-B promoter. ERRs constitutively stimulate transcriptional activity of genes in the absence of ligand (25, 26). This has been supported by recent studies on ERR{alpha} (16) and ERR{gamma} (56) in which LBD crystal structures revealed a classical agoinst conformation. The stimulation of the MAO-B promoter by ERRs is dependent on the regions that contain proximal and distal AGGTCA motifs (Fig. 4Go), which are the core of estrogen-related receptor response element, ERRE (18, 57). This finding is consistent with observations that the multiple ERREs constitute a key regulatory unit for ERR action (23, 58, 59). Although ERRs bind preferentially to ERRE, they also bind a variety of EREs and ER coactivators (57). Because both AF2 and DBD of the ERRs are, to a large extent, involved in the transactivation of the MAO-B promoter and because ER{alpha} lacking either AF2 or the DBD domain loses the ability to repress ERRs, we suggest that binding to the DNA element as well as interactions with coactivators or corepressors are involved in the cross talk between the ERRs and ERs.

Based on the competitive nature of ERRs and ERs in binding to response elements, coactivators, and selective ligands, ERRs were found as a modulator of ER-mediated transactivation (19, 20, 26). However, on the MAO-B promoter, ERs inhibit the transactivation function of ERRs without activating the promoter. To further complicate the observations, ER{alpha} and ERß may have different mechanisms in repressing the ERR-stimulated activity because E2-dependent ER{alpha} repression could be reversed by ICI but not by ERß-mediated E2 action. Additionally, ERß showed a much stronger inhibition of ERR{gamma}-induced activity than that shown with ERR{alpha}. In agreement with these findings, ERß was detected at all the AGGTCA-containing regions, but ER{alpha} was detected in only one region of the MAO-B promoter (Fig. 7Go, B and F). In addition, with the overexpression of ER{alpha} in HeLa cells, it only displaces the endogenous ERRs from the proximal AGGTGACCT region, whereas ERß is able to displace ERRs from all regions (Fig. 7CGo). These results were not totally unexpected, because isoforms of ERRs and ERs have been shown to possess differences in promoter preference and biological function (3, 60, 61). ERß is highly expressed in the nervous tissue (62) and may be expected to have a greater effect on MAO-B expression than ER{alpha}.

When ERs bind to an agonist, they assume a conformation that favors coactivator recruitment and results in gene activation. When binding to an antagonist, the ER conformation changes in favor of interaction with corepressors and results in gene repression (reviewed in Ref. 63 ; see references therein). However, estrogen-dependent repression of gene activity is a common occurrence (1). Several ER corepressors were identified (64, 65, 66, 67), and it is not clear whether these corepressors are involved in MAO-B repression. In the presence of E2 and ERs, however, we did detect an increase of mSin3A, a component of the histone deacetylase complex, and nuclear receptor corepressors, SMRT and RIP140 (61), at regions of the MAO-B chromatin that are responsible for stimulation by ERRs. On the other hand, both histone 3 and 4 acetylation and the amount of CBP at these regions were decreased (Fig. 6Go, D and E). Our data support a common mechanism of ER-mediated repression by recruiting chromatin-modifying proteins and forming a repressed chromatin state (64, 65, 66). However, it is not clear why agonist-bound ERs recruit corepressors to the MAO-B promoter. Other than ligand, the organization of the estrogen response unit that contains a variety of response elements of the promoter is influencing the conformation of the bound receptor, dictating the interaction with cofactors and, consequently, different response outcomes (27, 61, 68, 69). For example, with respect to the lactoferrin gene promoter, the estrogen response differs between the human or mouse in mammalian gland cells even though both promoters have a near identical estrogen response element at a similar location (27). Therefore, native chromatin structure and the arrangement of the natural gene promoter are critical factors in determining receptor binding and response to a stimulus. The MAO-B promoter was not as responsive to ERR stimulation in ER-positive cells, such as MCF-7 and T47D (Fig. 6Go) as in ER-negative cells, such as HeLa. One possible explanation for the differential response of MAO-B promoter to ERRs in different cell lines is that the high endogenous ERs in the MCF-7 and T47D cells may interfere with binding of ERRs to the MAO-B promoter and suppress the promoter activity. Consistent with this hypothesis, addition of E2 further reduced the ERR-stimulated MAO-B promoter activity in MCF-7 cells. These in vivo data add more support to the interplay between the ERs and ERRs and the physiological relevance in regulation of MAO-B gene expression. This observation is also in agreement with the finding that ERR{alpha} activates the ERE reporter in ER-negative cell lines and represses the ERE reporter in ER-positive cell lines (48).

Recent studies on energy expenditure showed that ERR{alpha} is a key player in the PGC-1{alpha}-regulated energy metabolism program (70, 71) as well as in genes involved in mitochondria biogenesis and oxidative phosphorylation pathway. These studies have expanded the function of ERR{alpha} from a modulator in estrogen pathway into a major player in regulation of fatty acid metabolism (72, 73) and lipid absorption (29). Our study showed that MAO-B is highly expressed in metabolically active tissues, which agrees with the pattern of ERR expression and functionally, with MAO-B as a target of ERRs. The present study supported the microarray study with MDA-MB-231 breast cancer cells in which overexpression of ERR{alpha} strongly induced MAO-A and MAO-B expression (13). These studies implicated an additional function of ERRs in a neurotransmitter pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Line and Reagents
HeLa (human cervical adenocarcinoma), MCF-7 (human mammary gland adenocarcinoma), T47D (human mammary gland ductal carcinoma) cells, MEM, Eagle’s medium, and RPMI 1640 were purchased from American Type Culture Collection (Manassas, VA), and fetal bovine serum was purchased from Sigma (St. Louis, MO). E2 was from Sigma and ICI was from TOCRIS (Ellisville, MO). 4-OHT was purchased from Research Biochemicals International (Natick, MA). A rabbit polyclonal antibody to the ERR{alpha} peptide at the C terminus (P3–80) or N terminus (P2–77) was purified with protein-A column as described previously (46). The epitope affinity-purified rabbit ERR{gamma} IgG was a gift from Affinity BioReagents, Inc. (Golden, CO). The rabbit polyclonal anti-ERR{gamma} antibody (pG676) raised against peptide from the N-terminal region (LYPSAPILGGSGPVRKLYDDCSS) from our laboratory was used in Western blot analysis. MAO-B antibody was previously described (74). Commercial mouse monoclonal antibodies were obtained from the following sources: ER{alpha} (TE111.5D11) from NeoMarker (Fremont, CA); ERß (clone 9.88, IgM) from Sigma; c-Myc (9E10) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and SMRT (1542) from Abcam, Inc. (Cambridge, MA). The rabbit polyclonal antibodies against acetylhistone H3 and acetylhistone H4 were from Upstate Biotechnology, Inc. (Charlottesville, VA), whereas mSin3A (K-20), CBP (A-20), and RIP-140 (H-300 and K-18) were from Santa Cruz Biotechnology. Purified normal rabbit IgG was from Sigma.

Plasmids
The human MAO-B promoter-luciferase reporter constructs (wt, D4, D5, and D10) (75) and expression vector of nuclear receptors ERR{alpha}, His-Myc-ERR{alpha} (59) were described previously. Mutations made at the potential ERR-binding sites of the MAO-B promoter were carried out with the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). The GG to AA mutation were made at –1762 (m1), –1468 (m2), and –289 (m3), and the CC to AA mutation was made at –286 (m4). The mouse mammary tumor virus-long terminal repeat-luciferase reporter construct that contains the GR response element was from Trevor K. Archer [NIHES/National Institutes of Health (NIH)], and the human GR expression plasmid was from R. Evans (Salk Institute, La Jolla, CA). Constructs of ERR{alpha} AF2 mutation (ERR{alpha} L413A/L418; AF2m) and P-box mutation (ERR{alpha} p-boxm) (48) were gifts from Janet E. Mertz (University of Wisconsin Medical School, Madison, WI). ERR{gamma}, ERR{gamma} AF2{Delta} (ERR{gamma} 449, deletion of the last nine amino acids of the AF2 domain), and Myc-ERR{gamma} (49) were gifts from U. Borgmeyer (University of Hamburg, Hamburg, Germany). wt ER{alpha} was from Donald McDonnell (Duke University Medical Center, Durham, NC), and the deletion mutants HE11, HE15, and HE19 were from Pierre Chambon (Institute de Génétique et de Biologie Moléculaire et Cellulaire, Collège de France, Illkirch, France). ERß was from Kenneth S. Korach (National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, NC) and coactivator PGC-1 {alpha} (76) from A. Kralli (Scripps Research Institute, La Jolla, CA).

Cell Culture, Transient Transfection, and Luciferase Assay
HeLa and MCF-7 cells were maintained in MEM, Eagle, and T47D in RPMI 1640 medium; the culture medium was supplemented with 100 U/ml penicillin, 10 µg/ml streptomycin, and 10% fetal bovine serum at 37 C under 5% CO2. A day before the experiment, medium supplemented with charcoal-stripped serum was used instead. Transfections were carried out with QIAGEN Effectene Transfection Reagent (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. DNA mixture consists of reporter constructs (100 ng per well), internal control (1 ng per well of Renilla luciferase pRL-CMV plasmid; Promega, Madison, WI), the nuclear receptor expression plasmids (specified in individual experiments), and the empty expression vector pSG5 to make the final amount of 201 ng. Before transfections, cells were plated in 24-well plates and grown overnight in medium containing 10% dextran-coated charcoal-stripped serum (Atlanta Biologicals, Norcross, GA). Cells were washed 4 h after transfection and fresh medium was added. In the ER transfection experiments, cells were treated with vehicle, 10–8 M 17ß-estradiol (E2), or E2 (10–8 M) plus ICI (10–6 M), whereas in the GR transfection experiments, 10–7 M DEX was used. After 24 h of continuing culture, cells were collected and the firefly and Renilla luciferase activities measured with Dual-Luciferase Reporter Assay System (Promega) on a Fluoroskan Ascent FL (Labsystems, Franklin, MA) instrument. The firefly luciferase reporter activities were normalized by Renilla luciferase activities and shown as relative light units (RLU). The data were the mean ± SD from a minimum of three independent experiments with duplicates for each experiment. Statistical analyses were performed with Student’s t test with P value < 0.05. The data are presented as fold of activation or percentage of activation, as specified in the individual figures.

Northern Blot Analysis
The MAO-A and -B mRNA distribution in human tissues was examined by using the FirstChoice Northern Human Blot I (Ambion, Inc., Austin, TX). The effects of ERR{alpha}, ERR{gamma}, or PGC-1{alpha} on MAO-B expression in HeLa cells were examined by transient transfection. ERR{alpha}, ERR{gamma}, or PGC-1{alpha} expression vector or pSG5 empty vector (2 µg) were transfected into HeLa cells for 24 h and the total RNA extracted with Qiagen RNeasy Mini Kit according to the supplier’s protocol (QIAGEN). Total RNA (10 µg) from the transfected cells was loaded onto each gel lane, electrophoresed, and transferred onto nylon membranes with the standard method. The human MAO-A or MAO-B (77), and mouse ß-actin probes were labeled with [{alpha}-32P]dCTP to the specific activities of more than 3 x 108 cpm/µg by Ready-To-Go DNA Labeling Beads (–dCTP) (Amersham Biosciences, Piscataway, NJ). Hybridization was carried out with NorthernMax (Ambion). Human tissue blot was hybridized overnight with the MAO-B probe and autoradiographed for 16 h. The blot was then stripped and rehybridized to the MAO-A probe overnight and autoradiographed for 3 h. The RNA blot of HeLa cells was hybridized with the MAO-B and ß-actin probe, and exposed for 48 and 2 h, respectively. The developed autoradiograms were scanned with a ChemiImager 5500 (Alpha Innotech Corp., San Leandro, CA) and quantified with AlphaEaseFC software. Intensity of the MAO-B band was normalized with the ß-actin band, and the control value was set as 1 arbitrary unit.

Western Blot Analysis
HeLa cells were transfected as described above, and cell lysate was prepared with lysis buffer. Protein concentration was determined by the bicinchoninic acid method (Pierce Chemical Co., Madison, WI), and a total of 40 µg protein was separated by 4–12% Bis-Tris NuPAGE gel. After electrophoresis, the proteins were electrotransferred to polyvinylidene difluoride membrane (NOVEX, San Diego, CA). Western blotting was carried out by specific antibody to MAO-B, ERR{alpha}, ERR{gamma}, ER{alpha}, ERß, and the ECL detection system (Amersham Biosciences). Blots were stripped according to the instruction of NOVEX and reprobed with specific antibody to ß-actin (mouse monoclonal antibody, SIGMARBI).

MAO-B Catalytic Activity Assay
The same protein sample from the Western blot analysis was used in the MAO-B enzyme assay (78). Total protein (100 µg) was incubated with 10 µM 14C-labeled phenylethylamine (Amersham Biosciences) in the assay buffer (50 mM sodium phosphate buffer, pH 7.4) at 37 C for 20 min, and the reactions were terminated by the addition of 100 µl of 6 N HCl. The products were then extracted with ethyl acetate/toluene (1:1) and centrifuged at 4 C for 10 min. The organic phase containing the reaction product was extracted, and its radioactivity was obtained by liquid scintillation spectrometry. Statistical analysis was performed with the Sigma State 3.1 software program.

ChIP Assay
The ChIP assay was performed according to the instructions of the ChIP Assay Kit (Upstate Biotechnology) with minor modifications. HeLa cells were transfected with 2 µg of empty vector or expression vectors of myc-ERR{alpha}, myc-ERR{gamma}, ER{alpha}, or ERß for 24 h. Proteins were cross-linked to the DNA by incubating the cells with 1% formaldehyde for 10 min at 37 C, and the cells were washed with cold PBS buffer twice before disruption in protease inhibitor cocktail (1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, and 1 µg/ml pepstatin A) containing sodium dodecyl sulfate lysis buffer. Chromatin was sonicated to an average DNA length of 200-1000 bp as verified by agarose gel electrophoresis. The sheared chromatin was then diluted in ChIP dilution buffer and evenly divided for input control and immunoprecipitation by specific antibody or normal rabbit IgG (5–10 µg). After addition of the antibodies, the chromatin solutions were gently rotated on a rotator overnight at 4 C. The protein A agarose slurry containing sonicated salmon sperm DNA was added to the antibody-bound chromatin solution and incubated for another hour at 4 C with constant rotation. The agarose beads were collected by centrifugation and washed, and the antibody-bound chromatin was released from the agarose beads according to the supplier’s specification. Finally, DNA was purified by phenol/chloroform extraction and ethanol precipitation. The MAO-B promoter/enhancer region was detected with PCR. The ChIP1 region (–438 to –278) was detected with the forward primer 5'-AGT AAT TGG GGC CCT GAA GGA-3' and the reverse primer 5'-GGG CGG AGA GGT CAC CTA GGA-3'; the amplicon is 161 bp; ChIP2 region (–1593 to –1404) forward primer, 5'-TCA CCT GGC ACG TAA TTC ACT-3'; reverse primer, 5'-CGA TCC CTA CCT CAT GTC C-3'; the amplicon is 190 bp; ChIP3 region (–1829 to –1693) forward primer, 5'-GCA AAG GCC TTC CCA ATA TGT-3'; reverse primer, 5'-TAG GTT CCA AGG GCT CCA TC-3'; the amplicon is 137 bp. The ChIP4 region is located more than 7 kb upstream from the promoter and served as negative control. The forward primer of ChIP4 is 5'-CAA CTA AAG GCA ACA TGT GAT-3'; reverse primer, 5'-GGC CCT CAA AGT CAG A-3'; the amplicon is 139 bp. The PCR conditions for ChIP assay were 94 C for 30 sec, 55 C for 30 sec, and 72 C for 30 sec, for a total of 35–40 cycles.


   ACKNOWLEDGMENTS
 
We thank J. E. Mertz, U. Borgmeyer, D. McDonnell, P. Chambon, K. Korach, A. Kralli, and R. Evans for providing reagents. We appreciate the comments and suggestions by J. Wachsman, B. Schrader, and A. Jetten. We thank J. Martin for editing the manuscript.


   FOOTNOTES
 
This work was supported by the Intramural Research Program of National Institutes of Health, National Institute of Environmental Health Sciences, and National Institute of Mental Health, R37 MH39085, MERIT Award, R01 MH67968, and the Boyd and Elsie Welin Professorship Award (to J.C.S.).

First Published Online February 16, 2006

Abbreviations: AF2, Activation function 2; CBP, cAMP response element-binding protein (CREB)-binding protein; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; DEX, dexamethasone; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; ERR, estrogen-related receptor; ERRE, estrogen-related receptor response element; GR, glucocorticoid receptor; ICI, ICI182,780; KO, knockout; LBD, ligand-binding domain; MAO, monoamine oxidase; 4-OHT, 4-hydroxytamoxifen; PGC, peroxisome proliferator-activated receptor coactivator; RIP140, receptor interacting protein 140; RLU, relative light unit; SMRT, silencing mediator of retinoid and thyroid hormone receptor; wt, wild type.

Received for publication June 27, 2005. Accepted for publication February 8, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   ERRα  |  ERRγ  |  GR
Coregulators:   RIP140  |  PGC-1  |  Sin3A  |  CBP  |  SMRT
Ligands:   Dexamethasone  |  17β-Estradiol  |  4-Hydroxytamoxifen



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