help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2003-0211
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kovacic, A.
Right arrow Articles by Clyne, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kovacic, A.
Right arrow Articles by Clyne, C. D.
Molecular Endocrinology 18 (1): 252-259
Copyright © 2004 by The Endocrine Society

Inhibition of Aromatase Transcription Via Promoter II by Short Heterodimer Partner in Human Preadipocytes

Agnes Kovacic, Caroline J. Speed, Evan R. Simpson and Colin D. Clyne

Prince Henry’s Institute of Medical Research (A.K., C.J.S., E.R.S., C.D.C.), Clayton, Victoria 3168, Australia; and Department of Biochemistry and Molecular Biology (A.K., E.R.S., C.D.C.), Monash University, Clayton Campus, Victoria 3800, Australia

Address all correspondence and requests for reprints to: Agnes Kovacic, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: agnes.kovacic{at}phimr.monash.edu.au.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen synthesis from C19 precursors is catalyzed by aromatase cytochrome P450. Overexpression of aromatase through atypical promoter usage (use of promoter II) in adipose tissue contributes to breast cancer development and progression. One tumor-derived factor that appears to contribute to this process is prostaglandin E2 (PGE2). A factor that regulates aromatase expression downstream of PGE2 is liver receptor homolog-1 (LRH-1). In a study of factors that inhibit LRH-1, we have examined the ability of short heterodimer partner (SHP) to inhibit aromatase transcription mediated by LRH-1 in preadipocytes. RT-PCR analysis indicated that both LRH-1 and SHP are expressed in human preadipocytes. To assess the effect of SHP on aromatase transcription, a transient transfection system was established using 3T3-L1 preadipocytes. Expression of SHP completely inhibited activity of an aromatase promoter II reporter gene induced by LRH-1. The combined treatment of forskolin and phorbol ester (which mimic PGE2) as well as LRH-1, which maximally induced reporter gene expression (140-fold), was also completely inhibited by SHP. This effect of SHP was mediated by inhibition of LRH-1 transcriptional activity, as measured by activity of GAL4-LRH-1 fusion constructs, and by inhibition of LRH-1 binding to promoter II. We conclude that SHP is a potent inhibitor of aromatase transcription in preadipocytes. Modulation of SHP expression and/or activity in adipose tissue may therefore have significant effects on aromatase expression and estrogen production in breast adipose tissue.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
APPROXIMATELY 70% OF breast cancers are estrogen receptor (ER) positive, and adjuvant antiestrogen therapy is thus an important treatment for these cancers. In postmenopausal women with breast cancer, the source of estrogen driving tumor growth is not circulating estrogen but rather that which is produced locally within the tumor and in the surrounding adipose tissue, due to activity of aromatase cytochrome P450 (1). We and others have shown that aromatase expression is elevated 3- to 4-fold within tumor-containing breast compared with normal breast (2, 3), as a result of epithelial-mesenchymal interactions that favor tumor growth (4). This may be one reason that aromatase inhibitors are superior to tamoxifen as first line treatment of advanced disease, in postmenopausal women (5). The main adverse effect of aromatase inhibitors, however, is that they inhibit estrogen production globally. Estrogen has neuroprotective effects and is important in maintaining bone homeostasis. It also has important roles in lipid homeostasis (6). Therefore, the inhibition of estrogen action in these areas has the potential to produce unfavorable side-effects such as osteoporosis, cognitive problems, and hepatic steatosis (7, 8, 9).

Aromatase is the product of the CYP19 gene, which in humans is expressed in many tissues including the granulosa and luteal cells of the ovary, bone, brain, placenta, testis, and adipose tissue (10). The structure of the CYP19 gene is complex: it spans 123 kb (11), with a coding region of 30 kb comprising 10 exons, of which exons II–X are translated into protein. The 5'-untranslated region is much larger and contains a number of alternative first exons that are spliced onto exon II in a tissue-specific manner. Thus, ovarian CYP19 transcripts contain untranslated exon II, whereas transcripts in adipose tissue contain untranslated exon I.4 (reviewed in Ref.12). Use of these alternative first exons is controlled by distinct upstream promoter sequences that are in turn regulated by unique mechanisms. For example, in the ovary, aromatase expression from the proximal promoter II is regulated by FSH, which acts through cAMP (13, 14). Placental aromatase expression is driven by the distal promoter I.1 and is under the control of retinoids (15). Adipose tissue expression of aromatase, by contrast, is driven by another distal promoter, promoter I.4, which is stimulated by glucocorticoids, class I cytokines, and TNF{alpha} (16, 17, 18).

Studies from several independent laboratories have shown that the increased expression of aromatase in breast cancer and adipose fibroblasts surrounding breast tumors occurs through a switch in promoter usage from promoter I.4 to promoter II (19, 20, 21). This appears to be the consequence of tumor-derived prostaglandin E2 (PGE2) stimulating promoter II activity via a pathway involving protein kinase A (PKA) and protein kinase C (PKC) (22). Because promoter II does not appear to be used in any other tissue of postmenopausal women, and local (rather than circulating) estrogens are the drivers of breast tumor growth in these patients, inhibition of CYP19 transcription from promoter II would represent a highly tissue-specific endocrine therapy that targets the estrogen required for tumor growth while sparing other important sites of estrogen action such as bone and brain. For this reason, we and others have studied the mechanisms that regulate promoter II in human breast cancer and adipose tissue as a first step toward the development of such selective aromatase modulators.

It has generally been believed that activity of promoter II in ovary (the main site of aromatase expression in premenopausal women) is dependent on the orphan nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) (14). Although adipose tissue does not express SF-1 (23) we recently showed that a related protein, liver receptor homolog-1 (LRH-1, NR5A2), is expressed in adipose tissue and substitutes for SF-1 as a competence factor for aromatase expression in this tissue (23) LRH-1 is coexpressed with aromatase in the stromal fraction of human adipose tissue and strongly activates transcription by binding to a nuclear receptor half-site within promoter II. LRH-1 is also overexpressed in primary breast tumors and surrounding adipose stroma, where its expression level correlates closely with that of aromatase (Suzuki, T., C. D. Clyne, R. Saito, Y. Miki, T. Ishida, T. Moriya, E. R. Simpson, and H. Sasano, manuscript submitted). It is likely, therefore, that LRH-1 is an important driver of aromatase expression in breast cancer, and consequently a candidate target for selective aromatase modulator development. The mechanisms that regulate LRH-1 expression and activity in adipose tissue are, however, poorly understood.

LRH-1 was first characterized as a liver-specific transcription factor, later shown to be involved in autoregulatory loops controlling cholesterol and bile acid homeostasis (24, 25). A key component of this hepatic system is the short heterodimer partner (SHP, NR0B2), an atypical nuclear receptor that lacks its own DNA binding domain and inhibits the action of other nuclear receptors (26), including LRH-1 (27). Our aim in the present study was to investigate whether SHP is expressed at appreciable levels in adipose tissue, and, if so, whether SHP can inhibit aromatase expression induced by LRH-1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LRH-1 and PKA/PKC Activate Promoter II Synergistically
Tumor-derived PGE2 is a potent stimulator of aromatase promoter II in preadipocytes (22). It acts by binding to EP2 and EP1 receptors linked to adenylyl cyclase and PKC, respectively, and its effects can therefore be mimicked by combined treatment with forskolin and phorbol ester (22). Because LRH-1 has recently been implicated as a key transcription factor mediating aromatase expression in breast cancer, we examined the possible interactions between these signaling pathways in the regulation of promoter II. 3T3-L1 preadipocytes were transfected with an LRH-1 expression vector in the presence or absence of forskolin and PMA (phorbol 12-myristate 13-acetate; phorbol ester). In the absence of forskolin and PMA, LRH-1 produced a 2-fold increase in the transcriptional activity of promoter II (Fig. 1Go). In the absence of exogenous LRH-1, forskolin and PMA increased promoter II activity to a similar extent (2-fold). In combination, however, LRH-1, forskolin, and PMA had a synergistic effect, increasing promoter II activity by 15-fold. This synergism cannot be attributed to forskolin and PMA stimulating endogenous LRH-1 expression as 3T3-L1 cells express very little to no LRH-1 (our unpublished observations).



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1. LRH-1 and PKA/PKC Regulate Aromatase Promoter II Synergistically

3T3-L1 mouse preadipocytes were cotransfected with a CYP19 promoter II-luciferase reporter construct (pII-516) in the presence and absence of an LRH-1 expression vector, and treated with or without forskolin (FSK, 25 µM) and phorbol ester (PMA, 4 nM). Luciferase activity is expressed as a ratio of ß-galactosidase activity. Similar results were obtained in two additional independent experiments.

 
Inhibitory Effect of SHP on LRH-1 Induced Promoter II Activity
To identify potential inhibitors of this marked induction of promoter II activity, we examined the effects of the nuclear receptor SHP. SHP binds to a variety of other nuclear receptors including ER, LXR, and LRH-1 and represses their transcriptional activity (26) and can also act as a direct transcriptional repressor. To assess the effect of SHP on LRH-1 induced CYP19 promoter II activity, 3T3-L1 cells were cotransfected with a promoter II reporter construct (pII-516), an LRH-1 expression construct (pCMX-LRH-1) and increasing concentrations of a SHP expression construct, pcDNA3.1+SHP (0.025–0.25 µg). Cells were then incubated in the presence or absence of forskolin and PMA for 8 h (Fig. 2Go). In the absence of forskolin and PMA, LRH-1 caused a 17-fold increase in promoter II activity compared with promoter II alone. This LRH-1 induced activity was dose dependently inhibited by increasing concentrations of SHP (Fig. 2Go). The highest dose of SHP inhibited promoter II activity by 85%. Treatment with forskolin and PMA in the presence of LRH-1 resulted in a 140-fold increase in promoter II activity. Again, this activity was potently and dose dependently inhibited by increasing concentrations of SHP, with the highest dose of SHP (0.25 µg) inhibiting luciferase activity by 96%.



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 2. SHP Dose Dependently Inhibits LRH-1 Induced Aromatase Promoter II Activity

3T3-L1 mouse preadipocytes were cotransfected with a CYP19 promoter II-luciferase reporter construct (pII-516), 0.5 µg LRH-1 expression vector (pCMX-mLRH-1) and increasing concentrations of a SHP expression vector, pcDNA3.1+-SHP (0, 0.025, 0.050, 0.075, 0.1, and 0.25 µg). This experiment was performed in the presence (right) or absence (left) of forskolin (FSK, 25 µM) and phorbol ester (PMA, 4 nM). Similar results were obtained in two additional independent experiments.

 
Inhibition of LRH-1 Activity by SHP
SHP has recently been reported to inhibit p300/CBP-mediated coactivation via the corepressor E1A-like inhibitor of differentiation 1 (EID1) (28). Therefore, part of the inhibitory effect of SHP on aromatase could arise through corepression of CREB-mediated transcription induced by cAMP (29), independent of LRH-1. To test this, 3T3-L1 preadipocytes were transfected with pII-516 in the presence or absence of SHP and forskolin/PMA, without exogenous LRH-1 (Fig. 3AGo). SHP did not inhibit basal activity of promoter II and produced a small but statistically insignificant reduction of forskolin/PMA induced activity, arguing against any LRH-1 independent inhibition of promoter II. Although 3T3-L1 cells express little or no endogenous LRH-1, this experiment was repeated using a promoter II reporter construct in which the LRH-1 binding site had been mutated, thus excluding any potential binding of endogenous LRH-1. Again, SHP failed to produced significant inhibition of either basal or stimulated promoter II activity. Thus, SHP does not inhibit aromatase promoter II in the absence of LRH-1.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3. SHP Dose Dependently Inhibits LRH-1 Transcriptional Activity

A, 3T3-L1 mouse preadipocytes were transfected with a CYP19 promoter II-luciferase reporter construct (pII-516) or a similar construct harboring a mutation in the LRH-1 binding site (pII-516mLRH), and an SHP expression vector (0.25 µg) or the empty parent vector pcDNA3.1+. Cells were then incubated in the presence (solid bars) or absence (open bars) of forskolin and PMA (25 µM and 4 nM, respectively) before being lysed for luciferase and ß-galactosidase activities. Data are the mean ± SD of average values from three independent experiments. Data are expressed as a percentage of control (pII-516 alone). B, 3T3-L1 mouse preadipocytes were cotransfected with the GAL4-LRH-1 fusion construct (G4-LRH-1) or the GAL4 DNA binding domain (G4), a GAL4 responsive luciferase reporter construct (g5-luciferase), and increasing concentrations of a SHP expression vector, pcDNA3.1+SHP (0, 0.025, 0.050, 0.075, 0.1, and 0.25 µg). Cells were then incubated in the presence (right) or absence (left) of forskolin and PMA (25 µM and 4 nM, respectively) before being lysed for luciferase and ß-galactosidase activities. Data are the mean ± SD of average values from three independent experiments. Data are expressed as a percentage of control (G4 alone).

 
To examine the direct effects of SHP on transcriptional activity of LRH-1, a GAL4-LRH-1 fusion construct was employed. The DNA binding domain and Ftz-F1 box of LRH-1 were replaced by the DNA binding domain of yeast GAL4. This construct was then cotransfected into 3T3-L1 cells along with a GAL4-responsive luciferase reporter as a reporter of LRH-1 transcriptional activity. SHP was able to dose dependently inhibit the transcriptional activity of LRH-1 with the highest dose of SHP (0.25 µg) resulting in a 60-fold reduction in LRH-1 transcriptional activity (Fig. 3BGo). Therefore the inhibition of aromatase by SHP is mediated by direct inhibition of LRH-1 transactivation. Because of the marked synergy between the PKA/PKC pathways and LRH-1 in activating aromatase promoter II (Fig. 1Go), we next asked if transcriptional activity of the GAL4-LRH-1 fusion construct is modified by these pathways, and if so, whether SHP can inhibit the transcriptional activity in the presence of forskolin/PMA. Transcriptional activity of LRH-1 was not increased in the presence of forskolin/PMA, and the inhibitory effects of SHP were similar in both treated and untreated cells.

Mechanism of Repression of LRH-1 by SHP
SHP has been shown to interact with the activation function-2 region of LRH-1 and compete for binding p160 coactivators such as SRC-3 (27). Once bound to its target and recruited to DNA, SHP also directly contributes to active transcriptional repression (27). The inhibitory effects of SHP on LRH-1-induced 12{alpha}-hydroxylase transcription occur through disruption of LRH-1 binding to DNA (30), whereas on other promoters (the shp promoter and synthetic response elements), SHP appears to exert its effects independently of ablation of LRH-1 DNA binding (27). To examine the effects of SHP on LRH-1 binding to aromatase promoter II, an EMSA was performed. A radiolabeled probe encompassing the promoter II LRH-1 response element was incubated with in vitro translated LRH-1 and increasing amounts of in vitro translated SHP (Fig. 4Go). Although unprogrammed reticulocyte extract formed nonspecific protein-DNA complexes (lane 2), addition of LRH-1 resulted in the formation of two additional protein DNA complexes (lane 3). Inclusion of SHP in the binding reaction dose dependently inhibited formation of these complexes (lanes 4–6), although SHP on its own did not bind the probe (lanes 7–9). The intensity of the upper (non-LRH-1) band associated with the in vitro translation reaction remained unchanged, thus eliminating the possibility of a non-specific protein competition effect. Quantification of the radioactivity associated with the LRH-1 complexes revealed that SHP inhibited binding of LRH-1 to promoter II by up to 80% (Fig. 4BGo).



View larger version (55K):
[in this window]
[in a new window]
  		Fig. 4. SHP Inhibits LRH-1 DNA Binding

A, In vitro transcribed/translated mouse LRH-1 (LRH-1, lanes 3–6) was incubated with radiolabelled probe encompassing the -130 AGGTCA motif (20,000 cpm) in the presence or absence of in vitro transcribed/translated mouse SHP (SHP, lanes 4–9). DNA/protein complexes were separated from free probe (lane 1) by gel electrophoresis and visualized by phosphorimaging. R.L., Reticulate lysate. B, Quantitation of the bands on the gel-shift revealed a 4-fold reduction in the ability of LRH-1 in vitro transcribed/translated protein to bind to the radiolabeled DNA probe.

 
Expression of Aromatase, LRH-1, and SHP in Human Adipose Tissue
To examine the expression profiles of these proteins in adipose tissue, semiquantitative RT-PCR was performed (Fig. 5Go). Consistent with previous studies, aromatase mRNA was detected only in adipose stromal cells that had been stimulated with forskolin or forskolin in the presence of PMA. This induction was mediated by promoter II, as evidenced by the parallel induction of aromatase using primers specific for exon II-containing transcripts. LRH-1 expression was also low in untreated cells but was significantly induced by treatment with forskolin in the presence or absence or PMA. SHP appeared to be constitutively expressed in all treatments, as was the control transcript, the 18S ribosomal subunit. Thus, human adipose stromal cells express both LRH-1 and SHP, both of which have the potential to modify aromatase expression and estrogen formation in breast.



View larger version (59K):
[in this window]
[in a new window]
  		Fig. 5. Aromatase and LRH-1 mRNA Levels Increase upon Stimulation with Forskolin and PMA

Semiquantitative RT-PCR analysis of human adipose stromal cells treated with either forskolin alone (FSK, 25 µM) or in combination with PMA (4 nM) revealed elevated expression of total aromatase transcripts as well as aromatase transcripts originating via the action of promoter II and LRH-1. SHP expression remained constant.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Aromatase inhibitors are now considered to be more effective than tamoxifen in first-line treatment of advanced breast cancer (5), as well as in the neoadjuvant setting (31). Whole-body aromatase inhibition has the potential, however, to accelerate bone loss and produce other symptoms of estrogen deficiency. This could be particularly significant in young postmenopausal patients or in women for whom prophylactic antiestrogen treatment is indicated. For these reasons, interest is growing in the concept of tissue-selective aromatase inhibitors. This prospect arises through the observations that local estrogen production in breast, rather than circulating estrogen, is the main driver of ER-positive tumor growth in postmenopausal women (12), and secondly that the promoter used to express aromatase in breast cancer (promoter II) is different from that used in other tissues in postmenopausal women. This appears to be the consequence of the action of tumor-derived factors, particularly PGE2, stimulating aromatase expression via promoter II. PGE2 acts via a downstream signaling pathway involving PKA and PKC (22). The combined treatment of factors that stimulate these pathways (forskolin/PMA) and LRH-1 results in a greater than additive effect on aromatase promoter II. At present, it is uncertain how this synergism occurs; however, LRH-1 may act as a basal factor and sensitize aromatase promoter II to the effects of CREB. A better understanding of the mechanisms that regulate aromatase promoter II and LRH-1 in the breast is therefore warranted.

SF-1 is a critical transcription factor required for promoter II-driven aromatase expression in the ovary. However, SF-1 is not present in breast adipose tissue. Instead, the closest homologue of SF-1, LRH-1 is present in the breast preadipocytes, as is aromatase. LRH-1 was recently shown to positively regulate aromatase expression via promoter II in breast adipose fibroblasts (23). This, taken with the fact that increased aromatase expression in adipose stroma surrounding breast tumors correlates with increased expression of LRH-1 (Suzuki, T., C. D. Clyne, R. Saito, Y. Miki, T. Ishida, T. Moriya, E. R. Simpson, and H. Sasano, manuscript submitted) suggests that this nuclear receptor could be a target for aromatase inhibition. Here we show that SHP potently inhibits promoter II activity in preadipocytes by inhibiting LRH-1 DNA binding and transcriptional activity. SHP is most closely related to another nuclear receptor, dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1; NROB1 (DAX-1) (32). Both SHP and DAX-1 lack a DNA binding domain and act as negative regulators of nuclear receptor function. Although most commonly associated with antagonism of SF-1 (33), DAX-1 also inhibits LRH-1 transcriptional activity (34). We were unable to detect DAX-1 expression in adipose tissue (data not shown); therefore, the present study focused on SHP, which we have shown to be present in human breast adipose (Fig. 5Go).

An important role for SHP in adipose tissue function was suggested by the observation that mutations that result in loss of SHP repressor activity are associated with mild obesity (35). Secondly, whereas SHP inhibits transcriptional activity of a range of nuclear receptors, it appears to enhance the transcriptional activity of peroxisomal proliferator-activated protein (PPAR) {gamma} in adipose tissue (36). This occurs via an atypical interaction with the DNA binding domain and hinge region of PPAR{gamma}, rather than at the activation function-2 domain of other receptors that are inhibited by SHP. Because ligands for PPAR{gamma} are very effective inhibitors of aromatase expression mediated by promoter II in adipose stromal cells (37), SHP would be expected to exert a dual inhibitory effect on aromatase expression; firstly by directly inhibiting the action of LRH-1 on promoter II, and secondly by potentiating the inhibitory action of PPAR{gamma} on this promoter.

SHP was first identified through an interaction screen using the constitutive androstane receptor as bait, but was also shown to interact with thyroid hormone and retinoic acid receptors (26). SHP also inhibits transcriptional activity of ligand-activated ER{alpha} (38) and ERß (32), as well as inhibiting the agonist activity of tamoxifen-bound ER in endometrial cancer cells (38). These data, taken with the inhibition of aromatase promoter activity shown here, suggest that SHP inhibits estrogen action at multiple levels. As such, induction of SHP expression or activity in breast would be relatively specific for inhibition of estrogen signaling. Little is known, however, regarding the regulation of SHP expression. The SHP promoter contains at least five nuclear receptor response elements that are bound by SF-1 in tissues in which both receptors are coexpressed, such as gonad and adrenal (39). Although adipose tissue does not express SF-1 (23), these elements can also be recognized by LRH-1 (39). Thus, SHP may inhibit its own expression via repression of LRH-1 activity in adipose. In liver, SHP expression is strongly induced by bile-acid bound farnesoid X receptor (FXR) (24, 25); however, the potential expression of FXR, or the effects of bile acids on SHP expression in adipose tissue, have not been studied. Regarding modulation of SHP activity, the receptor contains a conserved ligand-binding domain, raising the possibility that its activity may be modulated by natural or synthetic ligands, although no such ligands have been identified to date.

In summary we have shown that SHP inhibits aromatase expression in breast preadipocytes by preventing LRH-1 transactivation of promoter II. Identification of factors that regulate expression or activity of LRH-1 or SHP may therefore allow selective inhibition of aromatase activity mediated by this promoter in breast cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
pII-516 is a CYP19 promoter II/luciferase construct (in the vector PGL3, Promega, Madison, WI), which contains -516/-17 nucleotides of the human CYP19 promoter II (40). The expression construct that encodes mouse LRH-1 (pCMX-LRH-1) was a generous gift from David Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX). The cDNA insert of pCMX-LRH-1 was subcloned into pcDNA3.1+ (Invitrogen, Carlsbad, CA) for use in in vitro transcription/translation reactions. pCMV-ßgal encodes ß-galactosidase and was used to correct for transfection efficiency. pBIND (Promega) encodes the DNA binding domain (amino acids 1–147) of yeast GAL4 (G4). The GAL4/LRH-1 fusion construct (pBIND-LRH{Delta}) was created by replacing the DNA binding domain and Ftz-F1 box of LRH-1 with that of the GAL4 DNA binding domain. PCR fidelity and correct reading frame of the plasmid were confirmed by sequencing. The SHP cDNA was amplified by RT-PCR and subcloned into pcDNA3.1+ for use in transfections and in vitro transcription/translation reactions.

Cell Culture and Transfection
Human adipose stromal cells were isolated by collagenase digestion as previously described (41). 3T3-L1 cells were cultured in DMEM with 10% fetal calf serum at a density of 20,000 cells/ml. Cells were transfected for 24 h with 2 µg of total DNA comprising 1.0 µg of reporter construct, 0.5 µg expression construct (or empty vector for control), 0.02 µg pCMV-ßgal and varying concentrations of SHP expression plasmid (0.025-0.25 µg) using Fugene6 transfection reagent (Roche, Indianapolis, IN). Cells were serum starved for 24 h before experimental procedures. Luciferase and ß-galactosidase activity of soluble cell extracts was measured using the Luciferase Assay System (Promega) and ß-Gal Reporter Gene Assay (Roche).

EMSA
Recombinant proteins were transcribed/translated using the TNT Quick-coupled transcription/translation system (Promega). Four microliters of LRH-1 protein alone or with increasing volumes of SHP protein were incubated with 50,000 cpm 32P-labeled double-stranded probe (containing the LRH-1 binding site in bold: 5'-GAC TCT ACC AAG GTC AGA AAT GCT-3') for 15 min at room temperature in binding buffer [20 mM HEPES (pH 8.0), 1 mM EDTA, 10% glycerol, 50 mM KCl, 50 µg/ml poly(deoxyinosine·deoxycytidine/deoxyinosine·deoxycytidine), 1 mg/ml BSA, 10 mM dithiothreitol]. The reaction mixtures were electrophoresed on a 5.4% polyacrylamide gel in 0.5x TBE [final concentrations 44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA (pH 8.0)] as a running buffer for 4 h at 200 V. Gels were dried for 1 h and radioactive complexes were visualized by phosphorimaging (STORM scanner, Molecular Dynamics Inc., Sunnyvale, CA).

RT-PCR
Total RNA was prepared from human adipose stromal cells using the QiaAMP RNA Blood Mini kit (QIAGEN, Chatsworth, CA). First strand cDNA synthesis using 1.0 µg total RNA was performed using [(avian myeloblastosis virus (AMV)] Reverse Transcriptase (Roche) primed by random hexamers. PCRs were carried out using the following primer sets (all 5'-3'): LRH-1 (sense, CTG ATA CTG GAA CTT TTG AA; antisense, CTT CAT TTG GTC ATC AAC CTT); SHP (sense, TGG CCC AAG ATG CTG TGA C; antisense, TCG GGG TTG AAG AGG ATG GT); CYP19 (sense, TTG GAA ATG CTG AAC CCG AT; antisense, CAG GAA TCT GCC GTG GGA GA); 18S (sense, CGG CTA CCA CAT CCA AGG AA; antisense, GCT GGA ATT ACC GCG GCT).


   FOOTNOTES
 
This work was supported by the Victorian Breast Cancer Research Consortium Inc.

Abbreviations: CREB, cAMP response element binding protein; CBP, CREB binding protein; DAX-1, dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1, NROB1; ER, estrogen receptor; LRH-1, liver receptor homolog-1; PGE2, prostaglandin E2; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PPAR, peroxisome proliferator-activated protein; SF-1, steroidogenic factor-1; SHP, short heterodimer partner.

Received for publication June 4, 2003. Accepted for publication October 21, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Simpson E, Rubin G, Clyne C, Robertson K, O’Donnell L, Jones M, Davis S 2000 The role of local estrogen biosynthesis in males and females. Trends Endocrinol Metab 11:184–188[CrossRef][Medline]
  2. Bulun SE, Price TM, Aitken J, Mahendroo MS, Simpson ER 1993 A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription. J Clin Endocrinol Metab 77:1622–1628[Abstract]
  3. Harada N 1997 Aberrant expression of aromatase in breast cancer tissues. J Steroid Biochem Mol Biol 61:175–184[CrossRef][Medline]
  4. Simpson E, Rubin G, Clyne C, Robertson K, O’Donnell L, Davis S, Jones M 1999 Local estrogen biosynthesis in males and females. Endocr Relat Cancer 6:131–137[Abstract]
  5. Mouridsen H, Gershanovich M, Sun Y, Perez-Carrion R, Boni C, Monnier A, Apffelstaedt J, Smith R, Sleeboom HP, Janicke F, Pluzanska A, Dank M, Becquart D, Bapsy PP, Salminen E, Snyder R, Lassus M, Verbeek JA, Staffler B, Chaudri-Ross HA, Dugan M 2001 Superior efficacy of letrozole versus tamoxifen as first-line therapy for postmenopausal women with advanced breast cancer: results of a phase III study of the International Letrozole Breast Cancer Group. J Clin Oncol 19:2596–2606[Abstract/Free Full Text]
  6. Jones ME, Thorburn AW, Britt KL, Hewitt KN, Wreford NG, Proietto J, Oz OK, Leury BJ, Robertson KM, Yao S, Simpson ER 2000 Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity. Proc Natl Acad Sci USA 97:12735–12740[Abstract/Free Full Text]
  7. Paganini-Hill A, Clark LJ 2000 Preliminary assessment of cognitive function in breast cancer patients treated with tamoxifen. Breast Cancer Res Treat 64:165–176[CrossRef][Medline]
  8. Murata Y, Ogawa Y, Saibara T, Nishioka A, Fujiwara Y, Fukumoto M, Inomata T, Enzan H, Onishi S, Yoshida S 2000 Unrecognized hepatic steatosis and non-alcoholic steatohepatitis in adjuvant tamoxifen for breast cancer patients. Oncol Rep 7:1299–1304[Medline]
  9. Pinto HC, Baptista A, Camilo ME, de Costa EB, Valente A, de Moura MC 1995 Tamoxifen-associated steatohepatitis—report of three cases. J Hepatol 23:95–97[CrossRef][Medline]
  10. Bulun SE, Simpson ER 1994 Regulation of aromatase expression in human tissues. Breast Cancer Res Treat 30:19–29[CrossRef][Medline]
  11. Sebastian S, Bulun SE 2001 A highly complex organization of the regulatory region of the human CYP19 (aromatase) gene revealed by the Human Genome Project. J Clin Endocrinol Metab 86:4600–4602[Free Full Text]
  12. Simpson ER, Clyne C, Rubin G, Boon WC, Robertson K, Britt K, Speed C, Jones M 2002 Aromatase—a brief overview. Annu Rev Physiol 64:93–127[CrossRef][Medline]
  13. Steinkampf MP, Mendelson CR, Simpson ER 1987 Regulation by follicle-stimulating hormone of the synthesis of aromatase cytochrome P-450 in human granulosa cells. Mol Endocrinol 1:465–471[Abstract/Free Full Text]
  14. Michael MD, Kilgore MW, Morohashi K, Simpson ER 1995 Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary. J Biol Chem 270:13561–13566[Abstract/Free Full Text]
  15. Sun T, Zhao Y, Mangelsdorf DJ, Simpson ER 1998 Characterization of a region upstream of exon I. 1 of the human CYP19 (aromatase) gene that mediates regulation by retinoids in human choriocarcinoma cells. Endocrinology 139:1684–1691[Abstract/Free Full Text]
  16. Zhao Y, Mendelson CR, Simpson ER 1995 Characterization of the sequences of the human CYP19 (aromatase) gene that mediate regulation by glucocorticoids in adipose stromal cells and fetal hepatocytes. Mol Endocrinol 9:340–349[Abstract/Free Full Text]
  17. Zhao Y, Nichols JE, Bulun SE, Mendelson CR, Simpson ER 1995 Aromatase P450 gene expression in human adipose tissue. J Biol Chem 270:16449–16457[Abstract/Free Full Text]
  18. Zhao Y, Nichols JE, Valdez R, Mendelson CR, Simpson ER 1996 Tumor necrosis factor-{alpha} stimulates aromatase gene expression in human adipose stromal cells through use of an activating protein-1 binding site upstream of promoter 1.4. Mol Endocrinol 10:1350–1357[Abstract/Free Full Text]
  19. Agarwal VR, Bulun SE, Leitch M, Rohrich R, Simpson ER 1996 Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients. J Clin Endocrinol Metab 81:3843–3849[Abstract/Free Full Text]
  20. Harada N, Utsumi T, Takagi Y 1993 Tissue-specific expression of the human aromatase cytochrome P-450 gene by alternative use of multiple exons 1 and promoters, and switching of tissue-specific exons 1 in carcinogenesis. Proc Natl Acad Sci USA 90:11312–11316[Abstract/Free Full Text]
  21. Zhou D, Zhou C, Chen S 1997 Gene regulation studies of aromatase expression in breast cancer and adipose stromal cells. J Steroid Biochem Mol Biol 61:273–280[CrossRef][Medline]
  22. Zhao Y, Agarwal VR, Mendelson CR, Simpson ER 1996 Estrogen biosynthesis proximal to a breast tumor is stimulated by PGE2 via cyclic AMP, leading to activation of promoter II of the CYP19 (aromatase) gene. Endocrinology 137:5739–5742[Abstract]
  23. Clyne CD, Speed CJ, Zhou J, Simpson ER 2002 Liver receptor homologue-1 (LRH-1) regulates expression of aromatase in preadipocytes. J Biol Chem 277:20591–20597[Abstract/Free Full Text]
  24. Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, Galardi C, Wilson JG, Lewis MC, Roth ME, Maloney PR, Willson TM, Kliewer SA 2000 A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 6:517–526[CrossRef][Medline]
  25. Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J, Mangelsdorf DJ 2000 Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol Cell 6:507–515[CrossRef][Medline]
  26. Seol W, Choi HS, Moore DD 1996 An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors. Science 272:1336–1339[Abstract]
  27. Lee YK, Moore DD 2002 Dual mechanisms for repression of the monomeric orphan receptor liver receptor homologous protein-1 by the orphan small heterodimer partner. J Biol Chem 277:2463–2467[Abstract/Free Full Text]
  28. Bavner A, Johansson L, Toresson G, Gustafsson JA, Treuter E 2002 A transcriptional inhibitor targeted by the atypical orphan nuclear receptor SHP. EMBO Rep 3:478–484[CrossRef][Medline]
  29. Sofi M, Young MJ, Papamakarios T, Simpson ER, Clyne CD 2003 Role of CRE-binding protein (CREB) in aromatase expression in breast adipose. Breast Cancer Res Treat 79:399–407[CrossRef][Medline]
  30. Castillo-Olivares A, Gil G 2001 Suppression of sterol 12{alpha}-hydroxylase transcription by the short heterodimer partner: insights into the repression mechanism. Nucleic Acids Res 29:4035–4042[Abstract/Free Full Text]
  31. Ellis MJ, Coop A, Singh B, Mauriac L, Llombert-Cussac A, Janicke F, Miller WR, Evans DB, Dugan M, Brady C, Quebe-Fehling E, Borgs M 2001 Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for ErbB-1- and/or ErbB-2-positive, estrogen receptor-positive primary breast cancer: evidence from a phase III randomized trial. J Clin Oncol 19:3808–3816[Abstract/Free Full Text]
  32. Johansson L, Thomsen JS, Damdimopoulos AE, Spyrou G, Gustafsson JA, Treuter E 1999 The orphan nuclear receptor SHP inhibits agonist-dependent transcriptional activity of estrogen receptors ER{alpha} and ERß. J Biol Chem 274:345–353[Abstract/Free Full Text]
  33. Ito M, Yu R, Jameson JL 1997 DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Mol Cell Biol 17:1476–1483[Abstract]
  34. Suzuki T, Kasahara M, Yoshioka H, Morohashi K, Umesono K 2003 LXXLL-related motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1. Mol Cell Biol 23:238–249[Abstract/Free Full Text]
  35. Nishigori H, Tomura H, Tonooka N, Kanamori M, Yamada S, Sho K, Inoue I, Kikuchi N, Onigata K, Kojima I, Kohama T, Yamagata K, Yang Q, Matsuzawa Y, Miki T, Seino S, Kim MY, Choi HS, Lee YK, Moore DD, Takeda J 2001 Mutations in the small heterodimer partner gene are associated with mild obesity in Japanese subjects. Proc Natl Acad Sci USA 98:575–580[Abstract/Free Full Text]
  36. Nishizawa H, Yamagata K, Shimomura I, Takahashi M, Kuriyama H, Kishida K, Hotta K, Nagaretani H, Maeda N, Matsuda M, Kihara S, Nakamura T, Nishigori H, Tomura H, Moore DD, Takeda J, Funahashi T, Matsuzawa Y 2002 Small heterodimer partner, an orphan nuclear receptor, augments peroxisome proliferator-activated receptor {gamma} transactivation. J Biol Chem 277:1586–1592[Abstract/Free Full Text]
  37. Rubin GL, Duong JH, Clyne CD, Speed CJ, Murata Y, Gong C, Simpson ER 2002 Ligands for the peroxisomal proliferator-activated receptor gamma and the retinoid X receptor inhibit aromatase cytochrome P450 (CYP19) expression mediated by promoter II in human breast adipose. Endocrinology 143:2863–2871[Abstract/Free Full Text]
  38. Seol W, Hanstein B, Brown M, Moore DD 1998 Inhibition of estrogen receptor action by the orphan receptor SHP (short heterodimer partner). Mol Endocrinol 12:1551–1557[Abstract/Free Full Text]
  39. Lee YK, Parker KL, Choi HS, Moore DD 1999 Activation of the promoter of the orphan receptor SHP by orphan receptors that bind DNA as monomers. J Biol Chem 274:20869–20873[Abstract/Free Full Text]
  40. Michael MD, Michael LF, Simpson ER 1997 A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 (CYP19) gene. Mol Cell Endocrinol 134:147–156[CrossRef][Medline]
  41. Ackerman GE, Smith ME, Mendelson CR, MacDonald PC, Simpson ER 1981 Aromatization of androstenedione by human adipose tissue stromal cells in monolayer culture. J Clin Endocrinol Metab 53:412–417[Abstract/Free Full Text]

NURSA Molecule Pages Link:

Nuclear Receptors:   SHP  |  LRH-1



This article has been cited by other articles:


Home page
 Cancer Res.Home page
L. Farhana, M. I. Dawson, M. Leid, L. Wang, D. D. Moore, G. Liu, Z. Xia, and J. A. Fontana
Adamantyl-Substituted Retinoid-Related Molecules Bind Small Heterodimer Partner and Modulate the Sin3A Repressor
Cancer Res., January 1, 2007; 67(1): 318 - 325.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. E. Swales, M. Korbonits, R. Carpenter, D. T. Walsh, T. D. Warner, and D. Bishop-Bailey
The farnesoid x receptor is expressed in breast cancer and regulates apoptosis and aromatase expression.
Cancer Res., October 15, 2006; 66(20): 10120 - 10126.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
I. Kijima, S. Phung, G. Hur, S.-L. Kwok, and S. Chen
Grape Seed Extract Is an Aromatase Inhibitor and a Suppressor of Aromatase Expression
Cancer Res., June 1, 2006; 66(11): 5960 - 5967.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
R. Safi, A. Kovacic, S. Gaillard, Y. Murata, E. R. Simpson, D. P. McDonnell, and C. D. Clyne
Coactivation of Liver Receptor Homologue-1 by Peroxisome Proliferator-Activated Receptor {gamma} Coactivator-1{alpha} on Aromatase Promoter II and Its Inhibition by Activated Retinoid X Receptor Suggest a Novel Target for Breast-Specific Antiestrogen Therapy
Cancer Res., December 15, 2005; 65(24): 11762 - 11770.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
S. E. Bulun, Z. Lin, G. Imir, S. Amin, M. Demura, B. Yilmaz, R. Martin, H. Utsunomiya, S. Thung, B. Gurates, et al.
Regulation of Aromatase Expression in Estrogen-Responsive Breast and Uterine Disease: From Bench to Treatment
Pharmacol. Rev., September 1, 2005; 57(3): 359 - 383.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
R. Kumar, A. E. Gururaj, R. K. Vadlamudi, and S. K. Rayala
The Clinical Relevance of Steroid Hormone Receptor Corepressors
Clin. Cancer Res., April 15, 2005; 11(8): 2822 - 2831.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kovacic, A.
Right arrow Articles by Clyne, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kovacic, A.
Right arrow Articles by Clyne, C. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals