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Endocrinology Vol. 143, No. 8 2863-2871
Copyright © 2002 by The Endocrine Society

ARTICLE

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

Gary L. Rubin1, Jenny H. Duong1, Colin D. Clyne, Caroline J. Speed, Yoko Murata, Changhong Gong and Evan R. Simpson

Victorian Breast Cancer Research Consortium Inc (G.L.R., C.D.C., C.J.S., Y.M., C.G., E.R.S.), Prince Henry’s Institute of Medical Research, Monash Medical Centre, Clayton, Victoria 3168, Australia; and Department of Biochemistry and Molecular Biology (G.L.R., J.H.D., C.D.C., C.J.S., E.R.S.), Monash University, Clayton, Victoria 3800, Australia

Address all correspondence and requests for reprints to: Evan R. Simpson, Ph.D., Prince Henry’s Institute of Medical Research, Level 4, Block E, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia. E-mail: . evan.simpson{at}med.monash.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Local estrogen biosynthesis in breast adipose tissue, catalyzed by P450 aromatase, contributes to the growth of breast carcinomas. Aromatase expression is regulated by a number of alternative promoters, and in normal adipose tissue it is primarily regulated via the distal promoter I.4. However, in breast adipose containing a tumor, aromatase expression is regulated by the proximal promoter II in response to tumor-derived factors. Previously we have shown that peroxisomal proliferator-activated receptor {gamma} (PPAR{gamma}) ligands inhibit aromatase expression in normal breast adipose tissue mediated by promoter I.4. In the present study, we investigated the effects of the PPAR{gamma} ligand troglitazone and the retinoid X receptor (RXR) ligand LG101305 on aromatase expression mediated by promoter II. In cultured human breast adipose stromal cells, troglitazone or LG101305 alone inhibited aromatase activity and expression stimulated by inducers of promoter II, in a concentration-dependent manner, and this inhibition was greater in the presence of both ligands. Reporter gene assays showed that troglitazone and LG101305 inhibit transcription from promoter II of the CYP19 gene. However, EMSAs showed that PPAR{gamma} and RXR{alpha} do not bind to promoter II of the CYP19 gene, indicating that PPAR{gamma}- and RXR-mediated inhibition of aromatase expression via promoter II occurs through an indirect mechanism of action. Because ligands for PPAR{gamma} and RXR inhibit aromatase expression in healthy breast adipose (via promoter I.4), as well as expression induced by tumor-derived factors (via promoter II), such compounds could find utility in the treatment of estrogen-dependent breast cancers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BREAST CANCER IS the most common form of cancer in Western women today. Postmenopausal women are more likely to develop breast cancer than younger women, and estrogens produced locally in breast adipose tissue appear to play an important role in cancer progression by stimulating proliferation of breast tumor cells, acting in a paracrine fashion (1, 2, 3, 4). Estrogen biosynthesis is catalyzed by the enzyme aromatase cytochrome P450 (for reviews see Refs. 5, 6). In humans, aromatase is encoded by the CYP19 gene, which is a member of the P450 superfamily of genes, which currently contains over 480 members in some 70 gene families (7, 8). Aromatase is responsible for catalyzing the aromatization of the A ring of C19 androgens to the phenolic A ring of C18 estrogens resulting in loss of the C19 angular methyl group as formic acid (5, 6, 9). In humans, aromatase is expressed in a variety of tissues including: ovary (10, 11); testis (12, 13); placenta (10); brain (14, 15); adipose tissue (1, 16); and osteoblasts of bone (17, 18). The human CYP19 gene spans at least 120 kb with a coding region of approximately 30 kb containing nine translated exons (II–X) (19, 20, 21, 22). Aromatase transcripts in the various tissue sites of expression contain different 5'-untranslated first exons due to the use of a number of alternative promoters that regulate aromatase expression in ovary (promoter II) (11), placenta (promoter I.1) (10, 23, 24) and adipose tissue (promoters I.4 and II) (25) via alternative splicing mechanisms (for reviews see Refs. 6 and 26). Each promoter is differentially regulated; in the ovary, expression via promoter II is under the control of FSH, whose actions are mediated by cAMP (27, 28), whereas promoter I.1 is regulated by retinoids (29), as well as by the hypoxia-inducible transcription factor, Mash-2 (30). Expression via the distal promoter I.4 in adipose tissue requires the synergistic actions of glucocorticoids and class I cytokines or TNF{alpha} (31, 32, 33). The former use a Janus-activated kinase 1/STAT3 (signal transducers and activators of transcription) signaling pathway in which the activated STAT3 binds to an interferon-{gamma} activating sequence element located upstream of promoter I.4 (32), whereas the latter may use an MAPK/AP-1 (activating protein 1) pathway resulting in binding of c-fos/c-jun to an AP1 site upstream of the interferon-{gamma} activating sequence element (33). While this is the case in normal adipose tissue, in breast adipose tissue containing a tumor, aromatase expression in the surrounding breast tissue is elevated as a result of promoter switching, whereby expression is regulated through the proximal promoter II. This appears to occur in response to tumor-derived factors produced by breast tumor fibroblasts and epithelium, as well as possibly macrophages recruited to the tumor site (34, 35, 36, 37). One such factor is likely to be prostaglandin E2 (PGE2), which binds to EP1 and EP2 receptor subtypes resulting in activation of both the protein kinase A and C pathways (38, 39, 40). In ovarian granulosa cells, protein kinase A phosphorylates and activates cAMP response element binding protein (CREB), which binds to a cAMP response element-like sequence upstream of promoter II (28). Together with binding of steroidogenic factor-1 (SF-1) to a hexameric half-site (AGGTCA), this results in activation of transcription of aromatase from promoter II (27). In the case of adipose tissue the combined stimulation of both protein kinase A and C pathways by either PGE2, or else forskolin plus phorbol esters results in maximal expression from promoter II (25, 38, 39, 40).

In adipose tissue, aromatase is primarily expressed in the mesenchymal stromal cells, rather than in mature adipocytes (41, 42). Consistent with this, we have previously demonstrated that ligands for the nuclear receptor PPAR{gamma} (peroxisomal proliferator-activated receptor-{gamma}), which plays a key role in adipogenesis (43, 44), inhibit aromatase expression in cultured breast adipose stromal cells via promoter I.4 (45). PPAR{gamma} is a member of the nuclear receptor superfamily, which also includes the retinoid X receptor (RXR), the thyroid hormone receptor, retinoic acid receptor, liver X receptor, and the vitamin D receptor. Ligands for PPAR{gamma} include the synthetic thiazolidinediones (TZDs) troglitazone and rosiglitazone, as well as 15d-PGJ2 (15-deoxy-{Delta} (12, 14)-Prostaglandin J2), proposed to be a naturally occurring ligand (for review, see Refs. 43 , 44 , 46 , 47). All of these ligands have been shown to inhibit aromatase expression via promoter I.4 of the CYP19 gene, whereas a metabolite of troglitazone, which was not a PPAR{gamma} ligand, had no effect on aromatase expression (45). Preliminary data also demonstrate that inhibition of aromatase expression via promoter I.4 by troglitazone is enhanced in the presence of an RXR ligand (48). Ligands for RXR, termed rexinoids, include the endogenous ligand 9-cis retinoic acid and the synthetic ligands LGD1069 and LG101305. PPAR{gamma}, as with most of the nonsteroid nuclear receptors, exerts its transcriptional effects as a heterodimer with RXR, in which binding of ligand to either receptor can activate transcription, but binding of ligands for both receptors simultaneously is more potent (49).

In this manuscript we show that the PPAR{gamma} ligand troglitazone and the RXR ligand LG101305 inhibit aromatase expression via promoter II of the CYP19 gene in human adipose stromal cells. These findings suggest that nuclear receptor ligands including TZDs and rexinoids might have utility in breast cancer therapy in postmenopausal women.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Adipose tissue was obtained from women undergoing reduction mammoplasty (Mr. Alan Kalus, Windsor, Australia), after receiving informed consent. Adipose stromal cells were isolated by collagenase digestion of adipose tissue as described (41) and maintained in primary culture at a density of 50,000 cells/ml in Weymouth’s enriched medium (Invitrogen, Sydney, New South Wales, Australia) supplemented with 15% fetal bovine serum (15% vol/vol) (Trace Biosciences, Melbourne, Victoria, Australia), and allowed to grow until confluent (5–6 d) before treatment. Following serum deprivation for 24 h, cells were treated for a further 24 h with 25 µM forskolin and 4 nM phorbol 12-myristate 13-acetate (PMA) (Sigma, Sydney, New South Wales, Australia) in the presence of either the PPAR{gamma} ligand, troglitazone (Parke-Davis, Ann Arbor, MI), the RXR ligand LG101305 (Ligand Pharmaceuticals, Inc., San Diego, CA), or both ligands at varying concentrations. Cells were either assayed for aromatase activity or harvested for total RNA.

Aromatase assay
Aromatase activity was determined after incubation of cells with [1ß-3H]androstenedione (NEN Life Science Products, Boston, MA) for 2 h and measured by the incorporation of tritium into [3H]water, as described previously (41).

Real-time PCR
Following treatment, total RNA was extracted from human breast adipose stromal cells using the RNeasy Mini Kit (QIAGEN, Melbourne, Victoria, Australia), according to manufacturer’s instructions. Aromatase expression was determined after 0.5 µg of total RNA was reverse transcribed employing AMV-reverse transcriptase (Roche Diagnostics Australia Pty. Ltd., Melbourne, Victoria, Australia) using random primers (Invitrogen). cDNAs were then quantitated by real-time PCR using a LightCycler (Roche) and the FastStart DNA Master SYBR Green I kit (Roche) as described by the manufacturer. Aromatase exon-specific primers (GeneWorks Pty. Ltd., Adelaide, SA, Australia) were as follows (all 5'-3'):

TTG GAA ATG CTG AAC CCG AT (5' end sense oligo from coding exon II)

GCA ACA GGA GCT ATA GAT (5' end sense oligo from promoter-II-specific sequence)

GTG ACC AAC TGG AGC CTG (5' end sense oligo from exon I.4)

CAG GAA TCT GCC GTG GGG AT (common 3' end antisense oligo from exon III).

Integrity of cDNA was checked by amplification of the ribosomal subunit 18S using the 18S-specific primers:

CGG CTA CCA CAT CCA AGG AA (5'-sense)

GCT GGA AAT ACC GCG GCT (3'-antisense).

Aromatase cDNA levels (arbitrary units) were determined from an aromatase cDNA standard curve (0.01–100 fg /µl) and normalized to 18S transcript levels.

Reporter gene constructs
A luciferase reporter gene construct: PII-516, containing 516 bp of the 5'-flanking sequence of promoter II of the human CYP19 gene in the pGL3 vector (Promega Corp., Melbourne, Victoria, Australia) was generated previously (27). The SF-1 site in PII-516 was mutated from AGGTCA to AttTCA by PCR-mediated mutagenesis to generate PII-516mSF-1.

Transient transfection and reporter gene assays
3T3-L1 cells, a mouse preadipocyte fibroblast cell line (ATCC no. CL-173) were cultured in DMEM supplemented with 10% fetal bovine serum at a density of 30,000 cells/ml. 3T3-L1 cells were transfected for 24 h with 1.5 µg of a luciferase reporter gene construct and cotransfected with 0.5 µg of pSV-ß-galactosidase control vector (Promega Corp.) using FuGENE 6 Transfection Reagent (Roche) according to manufacturer’s instructions. Following serum starvation, cells were stimulated with 25 µM forskolin and 4 nM PMA for 8 h and in the presence of either the PPAR{gamma} ligand troglitazone or the RXR ligand LG101305. Cells were then lysed and relative luciferase activity was measured (Promega Corp.) and normalized to ß-galactosidase activity (Tropix, Bedford, MA).

EMSA
Five nanograms of the PII-516 reporter gene construct was used as a template to generate two overlapping sequences (PII-524/-254 and PII-285/-9) of the 5'-flanking region of promoter II of the CYP19 gene by PCR using Taq DNA polymerase and the primer pairs (GeneWorks): (-524CAT TGT CGA CAC TAG AGA TGG CCT GA-499)/ (-254TAA ACT GCA GTC CAG AGG TGG AGT CAT-280) and ( -85ACA AGT CGA CTC CAC CTC TGG AAT GA-259)/ (-9ACA GCT GCA GTT ACT GTT TTA TAA TGT GAT CAG-41). The PCR products were purified using the QIAEX II DNA extraction kit (QIAGEN). Five hundred nanograms of PII-524/-254, PII-285/-9 and a double-stranded oligonucleotide containing a PPAR{gamma} response element (PPRE) (TCG AAA CTA GGT CAA AGG TCA TGT) were 32P-labeled with [{alpha}-32P]deoxy-CTP (Amersham Pharmacia Biotech) by Klenow polymerase (Roche) and used as probes to determine binding of PPAR{gamma}2 and RXR{alpha} synthesized by coupled in vitro transcription/translation from the expression constructs pSV SPORT-PPAR{gamma}2 (a generous gift from Dr. Bruce Spiegelman, Dana Farber Cancer Institute, Boston, MA) and pCMX-RXR{alpha} (a generous gift from Dr. Steve McKnight, University of Texas SouthWestern Medical Center, Dallas, TX) by SP6 RNA polymerase (Promega Corp.) and T7 polymerase (Promega Corp.), respectively, using the TNT Coupled Reticulocyte Lysate System (Promega Corp.). In vitro translated (IVT) proteins (2–4 µl) and radiolabeled probes (20,000 cpm) were incubated at room temperature for 20 min in 20-µl binding reactions (20 mM HEPES, pH 8.0; 50 mM KCl; 1 mM EDTA; 10% glycerol; 1 mM dithiothreitol; 0.5 mg/ml BSA; 0.05 mg/ml poly deoxyinosine-deoxycytidine) with or without antibody against either PPAR{gamma} or RXR{alpha} (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). IVT proteins and antibodies (1 µl) were preincubated on ice for 20 min before addition of probe. Protein/DNA complexes were resolved by electrophoresis on either 4% or 6% nondenaturing polyacrylamide gels in 0.5x Tris-borate EDTA. Gels were dried and visualized by PhosphorImaging (Amersham Pharmacia Biotech Pty. Ltd., Sydney, New South Wales, Australia).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The PPAR{gamma} ligand troglitazone and the RXR ligand LG101305 inhibit aromatase activity in human breast adipose stromal cells via promoter II
The basal level of aromatase activity in cultured human breast adipose stromal cells is low but can be up-regulated via the proximal promoter II by agents that stimulate cAMP production such as forskolin and dibutyryl cAMP (Fig. 1Go) (38, 39, 40). This effect is markedly potentiated in the presence of phorbol esters such as PMA (Fig. 1Go) (38, 39, 40), to levels similar to those seen with PGE2 (40). Treatment of cultured human breast adipose stromal cells with the PPAR{gamma} ligand troglitazone (Fig. 2AGo) or the RXR ligand LG101305 (Fig. 2BGo) demonstrated that these ligands inhibited forskolin and PMA-stimulated aromatase activity in a concentration-dependent manner. LG101305 and troglitazone inhibited aromatase activity with similar efficacy yielding IC50 values of 0.53 µM and 0.29 µM, respectively. Concentrations of 10 µM almost completely abolished aromatase activity.



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  		Figure 1. Stimulation of aromatase activity by inducers of promoter II in human breast adipose stromal cells. Aromatase activity in cultured human breast adipose stromal cells was stimulated for 24 h with 25 µM forskolin (FSK) or 1 mM dibutyryl cAMP with or without 4 nM PMA. Aromatase activity (pmol/mg protein·2 h) ± SEM was determined following a 2-h incubation with the aromatase substrate [1ß-3H]androstenedione (150 nM). Each treatment was performed in triplicate and each experiment repeated three times.

 


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  		Figure 2. Ligands for PPAR{gamma} and RXR inhibit forskolin- and PMA-induced aromatase activity in human breast adipose stromal cells. Human breast adipose stromal cells were treated for 24 h with 25 µM forskolin (FSK) and 4 nM PMA plus either: troglitazone alone (A); LG101305 alone (B); or troglitazone plus 10 µM LG101305 (C). Aromatase activity (pmol/mg protein·2 h) was determined following a 2-h incubation with the aromatase substrate [1ß-3H]androstenedione (150 nM) and expressed as a percentage of the positive control, FSK/PMA. Each treatment was performed in triplicate and each experiment repeated three times. Results are expressed as the mean of three experiments ± SEM.

 
Because PPAR{gamma} action requires heterodimerization with RXR, we investigated the combined effects of troglitazone and LG101305 on aromatase activity. When increasing concentrations of troglitazone were added in the presence of 10 µM of LG101305 (Fig. 2CGo), a further increase in inhibition of aromatase activity was observed. This result indicates that the inhibitory effect of troglitazone on aromatase activity is enhanced in the presence of LG101305.

Troglitazone and LG101305 inhibit aromatase expression in the presence of forskolin plus PMA in human breast adipose stromal cells
RNA was extracted from human breast adipose stromal cells treated with troglitazone or LG101305 in the presence of forskolin and PMA. RNA was reverse transcribed and the corresponding cDNA was amplified using primers against the coding region of the CYP19 gene to quantitatively measure aromatase expression by means of real-time PCR. Aromatase expression was quantified from a standard curve generated from known quantities of aromatase cDNA (data not shown) and normalized to 18S levels, determined from an 18S standard curve (data not shown). In the absence of treatment, the basal level of aromatase expression was very low, but was markedly up-regulated approximately 45-fold in response to forskolin and PMA (Fig. 3Go). Combined treatment with troglitazone and LG101305 (both 1 µM) inhibited aromatase expression by approximately 75%. To confirm that these treatments specifically modified promoter II-derived aromatase transcripts we performed exon-specific PCR. Promoter II-derived transcripts were undetectable under basal conditions but were strongly up-regulated in the presence of forskolin and PMA, to levels similar to those observed for total aromatase transcripts. Troglitazone and LG101305 inhibited this induction by approximately 75%. Exon I.4-containing transcripts were undetectable under any conditions. Thus, the induction of aromatase transcripts in response to forskolin and PMA is completely attributable to activation of promoter II. Further, the inhibitory effect of troglitazone and LG101305 on forskolin- and PMA-induced aromatase expression is also specific for promoter II.



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  		Figure 3. Troglitazone and LG101305 inhibit aromatase expression via promoter II in human breast adipose stromal cells. Total RNA was extracted from human breast adipose stromal cells treated for 24 h with 25 µM forskolin (FSK) and 4 nM PMA in the presence or absence of troglitazone and LG101305 (both 1 µM), and reverse transcribed. cDNAs were then used in real-time PCR using the LightCycler FastStart DNA Master SYBR Green I kit (Roche) to quantitatively measure aromatase expression. Primers specific for either total aromatase (coding region), exon II- or exon I.4-containing transcripts were used. Integrity of cDNA was checked by amplification of the ribosomal subunit 18S. Aromatase transcript levels (arbitrary units) were determined from a standard curve generated from known quantities of aromatase cDNAs (not shown) and normalized to 18S expression determined from an 18S standard curve (not shown). Results are expressed as the mean of three experiments ± SEM. *, Not detectable.

 
PPAR{gamma} and RXR ligands inhibit transcription from promoter II of the CYP19
To assess whether the mechanism of inhibition by PPAR{gamma} ligands and RXR ligands was a consequence of their ability to interfere with transcription of the CYP19 gene from promoter II, 3T3-L1 cells, a mouse preadipocyte cell line, were transfected with a luciferase reporter gene construct containing 516 bp of the 5'-flanking sequence of promoter II of the human CYP19 gene in the pGL3 vector (PII-516), as depicted in Fig. 4Go. The activity of PII-516 is stimulated approximately 3-fold with forskolin and PMA, which up-regulate aromatase expression through promoter II (Fig. 4Go). On addition of increasing concentrations of either troglitazone (Fig. 4AGo) or LG101305 (Fig. 4BGo), reporter gene activity was inhibited in a concentration-dependent manner. This suggests that the mechanism whereby PPAR{gamma} and RXR inhibit aromatase expression is via inhibition of promoter II activity. Again, the concentrations of troglitazone and LG101305 were similar to those that inhibited aromatase activity (Fig. 2Go) and expression (Fig. 3Go).



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  		Figure 4. Troglitazone and LG101305 inhibit transcription from the CYP19 gene promoter II. 3T3-L1 cells were transfected for 24 h with 1.5 µg of a luciferase reporter gene construct containing 516 bp of the 5'-flanking sequence of promoter II of the human CYP19 gene, PII-516 and cotransfected with 0.5 µg of pSV-ß-galactosidase control vector. Following serum starvation, cells were stimulated with 25 µM forskolin and 4 nM PMA for 8 h with or without the PPAR{gamma} ligand troglitazone (A) or the RXR ligand LG101305 (B). Cells were then lysed and relative luciferase activity was measured and normalized to ß-galactosidase activity. Results are expressed as the mean of three experiments ± SEM.

 
PPAR{gamma}-mediated inhibition of promoter II of the CYP19 gene is not a consequence of the binding of PPAR{gamma} to the nuclear receptor half-site upstream of promoter II
The 5'-flanking sequence of promoter II contains a single hexameric element AGGTCA located 130 bp upstream of the start site of transcription, which is identical with a nuclear receptor half-site (Fig. 5Go). In ovarian granulosa cells, this is the site that binds SF-1 (27). This site, and the region immediately downstream, have been shown to be inhibitory of aromatase expression in adipose stromal cells, by acting as a silencer element (S1) (50). To test whether this sequence may represent a potential binding site through which the inhibitory effects of PPAR{gamma} on the CYP19 gene promoter II are mediated, the AGGTCA sequence was mutated to AttTCA in the PII-516mSF-1 reporter construct (Fig. 6Go). Mutation of this site prevents the binding of SF-1 to promoter II (data not shown). 3T3-L1 cells were transfected with PII-516 (Fig. 6AGo) and the PII-516mSF-1 mutated construct (Fig. 6BGo). Reporter gene activity of PII-516 was stimulated after treatment with forskolin and PMA, and this activity was inhibited to basal levels of transcription by the addition of 10 µM troglitazone (Fig. 6AGo), similar to the results in Fig. 4AGo. However, when the reporter gene activity of the mutated construct, PII-516mSF-1 was measured after treatment with forskolin and PMA plus 10 µM troglitazone, this activity was also inhibited to basal levels (Fig. 6BGo). This result indicates that the inhibition of promoter II expression by troglitazone is not mediated through binding of PPAR{gamma} to this site.



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  		Figure 5. Sequence of the 5'-flanking region of promoter II of the CYP19 gene. The identified regulatory elements: CRE (cAMP response element); SF-1; AP-1; S1; and C/EBP (CCAAT/ enhancer binding protein) binding sites are depicted. This sequence has been published (10 19 ) and is assigned GenBank/European Molecular Biology Laboratory data bank accession no. J05105.

 


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  		Figure 6. Mutation of the nuclear receptor half-site upstream of promoter II of the CYP19 gene does not alleviate the inhibition of promoter II by troglitazone. 3T3-L1 cells were transfected for 24 h with 1 µg of a luciferase reporter gene construct containing either the 516 bp of the 5'-flanking sequence of promoter II of the human CYP19 gene, PII-516 (A), or the construct PII-516mSF-1 (B), where the SF-1 site in PII-516 was mutated from AGGTCA to AttTCA. Following serum starvation, cells were stimulated with 25 µM forskolin (FSK) and 4 nM PMA ± 10 µM troglitazone for 6 h. Cells were then lysed and relative luciferase activity was measured and normalized to protein levels. Results are expressed as the mean of three experiments ± SEM.

 
This observation was further supported when nuclear extract (NE) prepared from human breast adipose stromal cells was incubated with a 32P-labeled probe derived from S1 (as depicted in Fig. 5Go). Figure 7Go shows the formation of several complexes, which were specifically removed in the presence of the unlabeled competitors, either S1 or a consensus SF-1 response element. However, antibodies against PPAR{gamma} (or RXR{alpha}, not shown) failed to supershift any of the complexes indicating that PPAR{gamma} does not bind to this sequence. Both antibodies recognized the PPAR{gamma}/RXR heterodimer on a consensus PPAR{gamma} response element (Fig 8AGo). An antibody against SF-1 also failed to supershift any of the complexes, suggesting that endogenous SF-1 is not binding to the probe.



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  		Figure 7. Antibodies against PPAR{gamma} and SF-1 do not displace the protein band in nuclear extracts from adipose stromal cells complexed to the nuclear receptor half-site of promoter II. Human adipose stromal cell nuclear protein (5 µg) was incubated with a 32P-labeled double-stranded oligonucleotide, S1 (PII-133/-104, as depicted in Fig. 5Go) in the absence (NE) or presence of 50- or 100-fold molar excess of unlabeled homologous competitor (S1) or a 50-fold molar excess of unlabeled SF-1 competitor (PII-117/-140, as depicted in Fig. 5Go). Polyclonal antibodies against PPAR{gamma} (6 µg) or SF-1 (2 µg) were added to the binding reaction. Protein/DNA complexes were resolved on a 6% nondenaturing polyacrylamide gel and visualized by PhosphorImaging. FP, Free probe. Arrows indicate the positions of the specific complexes.

 


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  		Figure 8. IVT PPAR{gamma} and RXR{alpha} do not bind to promoter II of the human CYP19 gene. A 32P-labeled double-stranded oligonucleotide containing a consensus PPRE (A) and 32P-labeled DNA fragments containing 5'-flanking sequences of promoter II: PII-524/-254 and PII-285/-9 (B), were incubated with IVT PPAR{gamma}2 (2 µl) and/or RXR{alpha} (2 µl) with (2 µg) or without antibody against either PPAR{gamma} or RXR{alpha}. Protein/DNA complexes were resolved on a 6% (A) or 4% (B) nondenaturing polyacrylamide gel and visualized by PhosphorImaging. FP, Free probe; RL, reticulocyte lysate.

 
In vitro translated PPAR{gamma}2 and RXR{alpha} do not bind to promoter II of the CYP19 gene
To further determine that the PPAR{gamma}- and RXR-mediated inhibition of aromatase is not a result of the binding of the PPAR{gamma}/RXR heterodimer to promoter II of the CYP19 gene to repress transcription, two overlapping fragments of the 5'-flanking sequence of promoter II were generated by PCR. These two fragments of the CYP19 gene promoter II, termed PII-524/-254 and PII-285/-9 were then used as 32P-labeled probes to determine whether IVT PPAR{gamma} and RXR{alpha} would bind to promoter II. A probe containing a consensus PPRE was used as a positive control for PPAR{gamma}/RXR{alpha} binding. The EMSA in Fig. 8AGo shows that IVT PPAR{gamma}2 and RXR{alpha} bind to the consensus PPRE strongly as a heterodimer. This complex was supershifted by an antibody to RXR{alpha} and formed a precipitating complex with an antibody against PPAR{gamma}. However, when IVT PPAR{gamma}2 and RXR{alpha} were incubated with the PII-524/-254 and PII-285/-9 probes, no binding of PPAR{gamma}2 or RXR{alpha} was evident (Fig. 8BGo). Evidence of nonspecific (NS) binding was apparent, but these NS bands were not supershifted by antibody to either PPAR{gamma} or RXR{alpha}. Therefore, PPAR{gamma}/RXR-mediated inhibition of aromatase expression via the human CYP19 gene promoter II is not a consequence of the PPAR{gamma}/RXR heterodimer binding directly to this region of promoter II, with resulting transcriptional repression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because of their ability to increase insulin sensitivity, ligands for the nuclear receptor PPAR{gamma} have been actively studied for their therapeutic utility in the treatment of type 2 diabetes. One of these, namely rosiglitazone, is in clinical use in the United States for this purpose (replacing troglitazone). It has been shown that PPAR{gamma} ligands can stimulate differentiation of a number of cell types including 3T3-L1 preadipocytes (43), but also colon cancer cells (51) and breast cancer cell lines (52), suggesting that PPAR{gamma} ligands could have therapeutic utility in the treatment of certain forms of cancer, including breast cancer. Previously, we have demonstrated that ligands for the nuclear receptor PPAR{gamma} inhibit aromatase expression and hence estrogen biosynthesis in human breast adipose stromal cells (45), namely the TZDs troglitazone and rosiglitazone, as well as the presumptive endogenous PPAR{gamma} ligand 15d-PGJ2. These compounds were shown to inhibit aromatase expression stimulated by glucocorticoids and class I cytokines, or TNF{alpha} via the CYP19 gene promoter I.4, which regulates aromatase expression in normal adipose tissue.

In breast adipose tissue containing a tumor, aromatase expression is stimulated through promoter II in response to tumor-derived factors such as PGE2, which stimulates cAMP production (34, 35, 36, 37). Therefore, we investigated whether ligands for PPAR{gamma} would also inhibit CYP19 gene expression via promoter II in cultured breast adipose stromal cells. Here we used forskolin and the phorbol ester PMA to mimic PGE2 action to stimulate aromatase expression in cultured breast adipose stromal cells through promoter II (Fig. 1Go). Forskolin/PMA-stimulated aromatase expression was inhibited in a concentration-dependent manner on treatment with the PPAR{gamma} ligand troglitazone or the RXR ligand LG101305 (Figs. 2Go and 3Go). This inhibition was further enhanced in the presence of both ligands (Fig. 2Go). One interesting point to note, was that troglitazone and LG101305 in combination inhibited aromatase activity from promoter II at concentrations that are an order of magnitude less than those which inhibit promoter I.4-mediated expression (45), although LG101305 alone inhibited aromatase activity from both promoters with similar efficacy (48). Both ligands inhibited the activity of a reporter gene construct regulated by promoter II of the CYP19 gene, suggesting that PPAR{gamma} and RXR interfere with transcription from promoter II (Fig. 4Go). One possible mechanism through which this inhibition could be mediated is via binding of the PPAR{gamma}/RXR heterodimer to the region including the hexameric nuclear receptor half-site 130 bp upstream of promoter II (Fig. 5Go), where SF-1 binds in granulosa cells (27). However, when this site was mutated, troglitazone still inhibited the luciferase activity of the mutated construct PII-516mSF-1 (Fig. 6Go), suggesting that the PPAR{gamma}/RXR heterodimer does not bind to this site. This was further confirmed with EMSA studies using nuclear extracts from breast adipose stromal cells (Fig. 7Go). Further EMSA studies were used to determine whether the PPAR{gamma}/RXR heterodimer binds to promoter II to directly repress transcription of the CYP19 gene from this promoter. Two regions of the 5'-flanking sequence of promoter II were used as probes to determine binding of IVT PPAR{gamma}2 and RXR{alpha}. However, recombinant PPAR{gamma}2 and RXR{alpha} did not bind to promoter II (Fig. 8Go), indicating that the mechanism of PPAR{gamma}-mediated inhibition of aromatase expression is not a consequence of the PPAR{gamma}/RXR heterodimer binding to promoter II of the CYP19 gene, but rather through an indirect mechanism of action. One possibility is that PPAR{gamma}/RXR competes for binding with nuclear cofactors such as CREB-binding protein, which plays a universal role in mediating the transcriptional responses of genes to multiple signaling pathways, and likely also is involved in regulation of aromatase expression via promoter I.4. Alternatively, another PPAR{gamma}-regulated gene may be mediating this effect. These possibilities are currently under investigation.

At the present time, first line therapies against estrogen-dependent breast cancer are aimed at targeting the mitogenic action of estrogen by blocking its action at the estrogen receptor or by blocking its synthesis from C19 precursors. Estrogen receptor antagonists such as tamoxifen and aromatase inhibitors such as Letrozole and Anastrozole are in use today in the treatment and management of breast cancers. In particular, aromatase inhibitors are now finding increasing use as first line therapy because their beneficial effects may exceed those of tamoxifen (53). However, aromatase inhibitors have a potential disadvantage in that they inhibit aromatase globally in all tissues where it is expressed such as bone and brain, leading to potential side effects such as loss of bone mineralization. Because aromatase is regulated in a tissue-specific fashion through the use of alternative promoters, the possibility of developing tissue-selective inhibitors of aromatase expression should have significant potential therapeutic benefit in the treatment and management of breast cancer.

The present findings suggest that nuclear receptor ligands including TZDs and rexinoids might have therapeutic utility in the management of breast cancer in postmenopausal women. Although ligands for PPAR{gamma} and RXR inhibit both basal aromatase expression (via promoter I.4), as well as expression induced by tumor-derived factors (via promoter II), these ligands show at least a 10-fold selectivity toward promoter II. The observation that in postmenopausal women, estrogens no longer serve as circulating hormones but rather are synthesized and act locally at extragonadal sites, together with the findings that tissue-specific aromatase expression is regulated by tissue-specific promoters, each with a different cohort of regulatory factors, allows for the development of the concept of selective aromatase modulators (54), the first generation of which could well be TZDs and/or rexinoids.


   Acknowledgments
 
The authors thank Parke-Davis (Ann Arbor, MI) for the generous gift of troglitazone, Drs. Veena Agarwal and Rich Heyman, formerly of Ligand Pharmaceuticals, Inc. (San Diego, CA) for LG101305, Dr. Bruce Spiegelman (Dana Farber Cancer Institute, Boston, MA) for pSV SPORT-PPAR{gamma}2, and Dr. Steve McKnight (University of Texas SouthWestern Medical Center, Dallas, TX), for pCMX-RXR{alpha}. We also thank Ms. Sue Panckridge for skilled graphical assistance.


   Footnotes
 
This work was supported by the Victorian Breast Cancer Research Consortium, Inc. (Anti-Cancer Council of Victoria, Australia) and by United States Public Health Service Grant R37-AG08174.

1 G.L.R. and J.H.D. contributed equally to this work. Back

Abbreviations: AP-1, Activating protein 1; CREB, cAMP response element binding protein; IVT, in vitro translated; NS, nonspecific binding; PGE2, prostaglandin E2; PMA, phorbol 12-myristate 13-acetate; PPAR{gamma}, peroxisomal proliferator-activated receptor {gamma}; PPRE, PPAR{gamma} response element; RXR, retinoid X receptor; S1, silencer element; SF-1, steroidogenic factor-1; TZDs, thiazolidinediones.

Received February 4, 2002.

Accepted for publication April 5, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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