Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M705409200 on December 14, 2007

J. Biol. Chem., Vol. 283, Issue 6, 3433-3444, February 8, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/6/3433    most recent
M705409200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Subbaramaiah, K.
Right arrow Articles by Dannenberg, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Subbaramaiah, K.
Right arrow Articles by Dannenberg, A. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

EP2 and EP4 Receptors Regulate Aromatase Expression in Human Adipocytes and Breast Cancer Cells

EVIDENCE OF A BRCA1 AND p300 EXCHANGE*Formula

Kotha Subbaramaiah{ddagger}1, Clifford Hudis§, Sung-Hee Chang, Timothy Hla, and Andrew J. Dannenberg{ddagger}

From the {ddagger}Department of Medicine, Weill Cornell Medical College, New York, New York 10065, the §Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10065 and the Center for Vascular Biology, Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3501

Received for publication, July 2, 2007 , and in revised form, December 13, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytochrome P450 aromatase (aromatase), a product of the CYP19 gene, catalyzes the synthesis of estrogens from androgens. Because aromatase-dependent estrogen biosynthesis has been linked to hormone-dependent breast carcinogenesis, it is important to elucidate the mechanisms that regulate CYP19 gene expression. The main objective of this study was to identify the receptors (EP) for prostaglandin E2 (PGE2) that mediate the induction of CYP19 transcription in human adipocytes and breast cancer cells. Treatment with PGE2 induced aromatase, an effect that was mimicked by either EP2 or EP4 agonists. Antagonists of EP2 or EP4 or small interference RNA-mediated down-regulation of these receptors suppressed PGE2-mediated induction of aromatase. PGE2 via EP2 and EP4 stimulated the cAMP->protein kinase A pathway resulting in enhanced interaction between P-CREB, p300, and the aromatase promoter I.3/II. Overexpressing a mutant form of p300 that lacks histone acetyltransferase activity suppressed PGE2-mediated induction of aromatase promoter activity. PGE2 via EP2 and EP4 also caused a reduction in both the amounts of BRCA1 and the interaction between BRCA1 and the aromatase promoter I.3/II. Activation of the aromatase promoter by PGE2 was suppressed by overexpressing wild-type BRCA1. Silencing of EP2 or EP4 also blocked PGE2-mediated induction of the progesterone receptor, a prototypic estrogen-response gene. In a mouse model, overexpressing COX-2 in the mammary gland, a known inducer of PGE2 synthesis, led to increased aromatase mRNA and activity and reduced amounts of BRCA1; these effects were reversed by knocking out EP2. Taken together, these results suggest that PGE2 via EP2 and EP4 activates the cAMP->PKA->CREB pathway leading to enhanced CYP19 transcription and increased aromatase activity. Reciprocal changes in the interaction between BRCA1, p300, and the aromatase promoter I.3/II contributed to the inductive effects of PGE2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen plays a significant role in the development and progression of breast cancer. Cytochrome P450 aromatase, encoded by the CYP19 gene, catalyzes the synthesis of estrogens from androgens (1). In postmenopausal women, peripheral aromatization in adipose tissue is largely responsible for estrogen production, and in particular mammary adipose tissue is considered an important local estrogen source. In breast cancers, aromatase activity is increased in both tumor and stromal cells (2), suggesting a role in tumor growth (3). In fact, estrogen deprivation is a commonly used approach for both the prevention and treatment of hormone-dependent breast cancer (4, 5).

Given the significance of estrogen synthesis in the pathogenesis of hormone-dependent breast cancer, it is important to elucidate the mechanisms that regulate the transcription of CYP19. The expression of CYP19 and thereby aromatase activity is regulated by several distinct promoters (6–8). In normal breast adipose tissue, the aromatase CYP19 gene is expressed at low levels under the control of promoter I.4, whereas increased expression occurs in breast cancers and cancer-proximal adipose tissue predominantly from promoters I.3 and II (7, 8). The proximal promoters I.3 and II are located close to each other and are activated by stimulation of the cAMP->protein kinase A (PKA)2->cAMP-responsive element-binding protein (CREB) pathway (9–12). Several findings suggest a key role for cyclooxygenase (COX)-derived prostaglandin E2 (PGE2) in stimulating CYP19 transcription via this pathway. PGE2 is a potent inducer of CYP19 transcription via a cAMP-dependent mechanism in vitro (9, 10, 13). Positive correlations have been detected between COX and aromatase expression in human breast cancer specimens (14–16). In mice genetically engineered to overexpress COX-2 in the mammary gland, increased levels of PGE2 and aromatase were found (17). Finally, use of aspirin, an inhibitor of COX-mediated PGE2 production, was associated with a reduced risk of hormone receptor-positive but not hormone receptor-negative breast cancer (18). Recently, the tumor suppressor, BRCA1, was found to negatively regulate CYP19 expression via selective inhibition of promoters 1.3 and II (19). This inhibitory effect of BRCA1 on aromatase activity was relieved by PGE2.


Figure 1
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 1.
EP2 is important for PGE2-mediated induction of aromatase in human adipocytes. A, cells were treated with the indicated concentration of PGE2; B–D, cells were treated with the indicated concentration of butaprost, an EP2 receptor agonist; E and F, cells were treated with vehicle, 500 nM PGE2, or 500 nM PGE2 plus the indicated concentration of AH6809, an EP2 receptor antagonist. All treatments were for 24 h. In A, B, and E, aromatase activity was determined as under "Experimental Procedures." Enzyme activity is expressed as femtomoles/µg of protein/min. Means ± S.D. are shown, n = 6. In A and B,*, p < 0.001 versus vehicle-treated cells; E,*, p < 0.01 versus PGE2-treated cells. In C, D, and F, total RNA was prepared. In C and F, 10 µg/lane RNA was subjected to Northern blotting. The blots were hybridized sequentially with the indicated probes. In D and F, aromatase mRNA was analyzed by real-time PCR as described under "Experimental Procedures." Values were normalized to the expression levels of glyceraldehyde-3-phosphate dehydrogenase. In D and F, means ± S.D. are shown, n = 3. D,*, p < 0.01 versus vehicle-treated cells and in F,*, p < 0.01 versus PGE2-treated cells.

 
Chronic use of selective COX-2 inhibitors, prototypic inhibitors of PG synthesis, has been associated with an increased risk of cardiovascular complications (20, 21). The mechanism underlying this toxicity is not fully understood, but inhibition of COX-2 results in the loss of all downstream PGs. It has been suggested that selective COX-2 inhibitors block the production of cardioprotective PGI2 by vascular endothelium, without inhibiting COX-1-dependent platelet thromboxane A2 synthesis, supporting a pro-thrombotic mechanism (22). To improve the therapeutic index, alternate treatment strategies are being explored. One possibility is to block the actions of PGE2 rather than to inhibit its production. PGE2 exerts its effects by binding to G protein-coupled receptors that contain seven transmembrane domains. Four subtypes of PGE2 receptor (EP1, EP2, EP3, and EP4) have been cloned and defined pharmacologically (23). Different EP receptors have been implicated in regulating cell proliferation, immune function, and angiogenesis (23, 24), but little is known about the regulation of aromatase. Hence, the primary objective of this study was to identify the EP receptor(s) that mediate the induction of aromatase by PGE2 in human adipocytes and breast cancer cells. We show that PGE2 via EP2 and EP4 activates the cAMP->PKA->CREB pathway resulting in enhanced CYP19 transcription and increased aromatase activity. This was due, at least in part, to reciprocal changes in the interaction between BRCA1, p300, and the aromatase promoter I.3/II.


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 2.
Induction of aromatase by PGE2 involves EP4. A–C, adipocytes were treated with the indicated concentration of PGE1 alcohol, an EP4 agonist; D and E, cells were treated with vehicle, 500 nM PGE2, or 500 nM PGE2 plus the indicated concentration of ONO AE3–208, an EP4 antagonist. F, cells were treated with vehicle, 1.0 µM butaprost, 0.2 µM PGE1 alcohol, butaprost plus 50 µM AH6809, butaprost plus 0.1 µM ONO AE3–208, PGE1 alcohol plus ONO AE3–208 or PGE1 alcohol plus AH6809 as indicated. All treatments were for 24 h. In A, D, and F, aromatase activity was determined as under "Experimental Procedures." Enzyme activity is expressed as femtomoles/µg of protein/min. Means ± S.D. are shown, n = 6. *, p < 0.01. In B, C, and E, total RNA was prepared and in B and E, 10 µg/lane was subjected to Northern blotting. The blots were hybridized sequentially with the indicated probes. In C and E, aromatase mRNA was analyzed by real-time PCR. Values were normalized to the expression levels of glyceraldehyde-3-phosphate dehydrogenase. In C and E, means ± S.D. are shown, n = 3. C, *, p < 0.01 versus vehicle-treated cells and in E, *, p < 0.01 versus PGE2-treated cells.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Adipocyte medium was purchased from ScienCellTM Research Laboratories (Vancouver, BC). Fetal bovine serum and TRIzol reagent were purchased from Invitrogen. PGE2, butaprost, PGE1 alcohol, and AH6809 were from Cayman Chemical Co. (Ann Arbor, MI). ONO (AE3–208) was a gift from ONO Pharmaceutical Co., Ltd. (Osaka, Japan). Rabbit polyclonal antisera for human phospho-CREB, BRCA1, p300, progesterone receptor (PR), β-actin, and control IgG were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Lowry protein assay kits, horseradish peroxidase-conjugated secondary antibody, glucose 6-phosphate, glycerol, pepstatin, leupeptin, glucose-6-phosphate dehydrogenase, and rotenone were from Sigma. A cAMP enzyme immunoassay kit was from Biomol (Plymouth Meeting, PA). PKA activity assay kits were from Calbiochem. ECL Western blotting detection reagents were from Amersham Biosciences. Nitrocellulose membranes were from Schleicher & Schuell (Keene, NH). 1β-[3H]Androstenedione and [32P]CTP were from PerkinElmer Life Sciences. Random-priming kits were from Roche Molecular Biochemicals (Indianapolis, IN). pSVβgal and plasmid DNA isolation kits were from Promega Corp. (Madison, WI). Luciferase assay reagents were from Analytical Luminescence (San Diego, CA). The CYP19 cDNA was obtained from Open Biosystem Inc (Huntsville, AL). The 18 S rRNA cDNA was purchased from Ambion, Inc. (Austin, TX). cDNAs for EP1–4 were from University of Missouri-Rolla Resource Center (Rolla, MO). siRNAs to EP1–EP4 and GFP were purchased from Dharmacon, Inc. (Lafayette, CO). Real-time PCR primers for PR were purchased from Applied Biosystems (Foster City, CA). Expression vectors for p300 and histone acetyltransferase activity mutant of p300 were from Upstate USA, Inc. (Charlottesville, VA). BRCA1 constructs were from Dr. Alvaro Monteiro (H. Lee Moffit Cancer Center, Tampa, FL) (25). The estrogen response element-luciferase construct was from Panomics (Fremont, CA).

Tissue Culture—Visceral adipocytes were obtained from ScienCellTM Research Laboratories. These primary cells were grown in adipocyte medium containing 10% fetal bovine serum. In experiments requiring transfection, cells were transiently transfected using a system from Amaxa (Gaithersburg, MD). SKBR3 cells were purchased from ATCC and grown in McCoy's 5a medium (modified) with 1.5 mM L-glutamine adjusted to contain 2.2 g/liter sodium bicarbonate, and 10% fetal bovine serum. Experimental treatments of adipocytes and SKBR3 cells were carried out under serum free conditions.


Figure 3
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 3.
EP2 and EP4 receptors mediate the induction of aromatase by PGE2. A, adipocytes were transfected with 2 µg of siRNAs to GFP or EP1–4 and allowed to grow for 36 h prior to analysis. In B, cells were transfected with 2 µg of siRNAs to GFP or EP1–4. 36 h later, cells were treated with vehicle (control) or 500 nM PGE2 for 24 h. In A and B, total RNA was prepared from cells and subjected to Northern blotting (10 µg/lane). The blots were hybridized sequentially with the indicated probes. In B, aromatase activity was determined in cell lysates as under "Experimental Procedures." Enzyme activity is expressed as femtomoles/µg of protein/min. Means ± S.D. are shown, n = 6. *, p < 0.01 versus PGE2-treated cells.

 
Animals—Generation of the mice used in this study has been described in detail previously (27). Briefly, MMTV-COX-2 transgenic mice on an FVB/N background were crossed with Ep2-/- mice in the C57BL/6J background. The resulting MMTV-COX-2 Ep2+/- mice in the (FVB/N and C57BL/6) background were backcrossed with FVB/N mice more than nine times. The resulting MMTV-COX-2 Ep2+/- female mice were crossed with Ep2+/- male mice in same strain yielding the different experimental groups. Mammary tissues were harvested, snap-frozen in liquid nitrogen and stored at -80 °C until analysis.

Northern Blotting—Prior to RNA extraction, frozen tissue was homogenized. Total RNA was prepared from mammary tissues and cell monolayers using an RNA isolation kit from Qiagen. 10 µg of total RNA/lane were electrophoresed in a formaldehyde-containing 1% agarose gel and transferred to nylon-supported membranes. EP1–EP4, BRCA1, aromatase and 18 S rRNA probes were labeled with [32P]CTP by random priming. The blots were probed as described previously (17). All experiments were repeated three times, and representative Northern blot data are shown.

Real-time Reverse Transcription-PCR—Total RNA was isolated using TRIzol reagent and reverse transcribed with the Superscript III First-Strand Synthesis System (Invitrogen). For total aromatase mRNA, the forward and reverse primers were 5'-CACATCCTCAATACCAGGTCC-3' and 5'-CAGAGATCCAGACTCGCATG-3', and the fluorescence-labeled probe was 5'-CCCTCATCTCCCACGGCAGATTCC-3'. For BRCA1, the forward and reverse primers were 5'-AAGAGGAACGGGCTTGGAA-3', 5'-AAAATAATCAAGAAGAGCAAAGCATGGATTCAAACTTA-3', and the fluorescence-labeled probe was 5'-CACACCCAGATGCTGCTTCA-3'. For glyceraldehyde-3-phosphate dehydrogenase mRNA (control), the forward and reverse primers were 5'-GAAGGTGAAGGTCGGAGTC-3' and 5'-GAAGATGGTGATGGGATTTC-3', and the fluorescence-labeled probe was 5'-CAAGCTTCCCGTTCTCAGCC-3'. Real-time PCR was carried out using an ABI Prism 7900 apparatus (Applied Biosystems).


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 4.
PGE2 via EP2 and EP4 stimulates the cAMP->PKA->CREB pathway resulting in induction of aromatase in adipocytes. A and B, adipocytes were transfected with 2 µg of siRNAs to GFP, EP2, or EP4. C, cells received 0.9 µg of siRNA to GFP, EP2, or EP4 and 0.9 µg of CYP19 promoter-luciferase. All cells also received 0.2 µg of pSVβgal. In A–C, 36 h after transfection, cells received fresh medium containing vehicle (control) or 500 nM PGE2. Treatments were for 24 h. Subsequently, cellular levels of cAMP (A) and PKA activity (B) were determined. Means ± S.D. are shown, n = 6. *, p < 0.01 versus PGE2-treated cells. In C, luciferase activity was measured in cell lysates, and the activities represent data that have been normalized to β-galactosidase activity. Means ± S.D. are shown, n = 6. *, p < 0.01 compared with PGE2 plus siRNA GFP. D, ChIP assays were performed. Top, cells were treated as indicated with vehicle (C), PGE2 (500 nM), butaprost (1 µM), or PGE1 alcohol (0.2 µM). Middle, cells were treated as indicated with vehicle, PGE2 (500 nM), PGE2 plus EP2 antagonist (50 µM AH6809), or PGE2 plus EP4 antagonist (0.1 µM ONO AE3–208). Bottom, cells were transfected with 2 µg of siRNAs to GFP, EP2, or EP4. 36 h after transfection, cells received fresh medium containing vehicle (C) or 500 nM PGE2. All treatments were for 2 h. Chromatin fragments were immunoprecipitated with antibodies against phospho-CREB, and the CYP19 I.3/II promoter was amplified by PCR (panel 1) or real-time PCR (panel 2). DNA sequencing was carried out, and the PCR product was confirmed to be the CYP19 I.3/II promoter. The CYP19 promoter was not detected when normal IgG was used or antibody was omitted from immunoprecipitation step (data not shown). For panel 2 (top, middle, and bottom), means ± S.D. are shown, n = 3. *, p < 0.01.

 
Transient Transfections—Cells were seeded at a density of 5 x 104 per well in six-well dishes and grown to 50–60% confluence. For each well, 2 µg of plasmid DNA was introduced into cells using 8 µg of Lipofectamine as per the manufacturer's instructions. After 24 h of incubation, the medium was replaced with basal medium. The activities of luciferase and β-galactosidase were measured in cellular extract.

Western Blotting—Lysates were prepared by treating cells with lysis buffer (150 mM NaCl, 100 mM Tris (pH 8.0), 1% Tween 20, 50 mM diethyldithiocarbamate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml trypsin inhibitor, and 10 µg/ml leupeptin). Lysates were sonicated for 20 s on ice and centrifuged at 10,000 x g for 10 min to sediment the particulate material. The protein concentration of the supernatant was measured by the method of Lowry et al. (28). SDS-PAGE was done under reducing conditions on 10% polyacrylamide gels as described by Laemmli (29). The resolved proteins were transferred onto nitrocellulose sheets as detailed by Towbin et al. (30). The nitrocellulose membrane was then incubated with primary antisera. Secondary antibody to IgG conjugated to horseradish peroxidase was used. The blot was probed with the ECL Western blot detection system according to the instructions of the manufacturer.

Measurements of cAMP Levels—Cells were plated at 5 x 104/well in six-well dishes and grown to 60% confluence before treatment. Amounts of cAMP were measured by enzyme immunoassay. Production of cAMP was normalized to protein concentration.

PKA Activity—Cells were plated at 5 x 104/well in six-well dishes and grown to 70% confluence before treatment. PKA activity was measured according to the instructions of the manufacturer. PKA activity was normalized to protein concentration.

Aromatase Assays—To determine aromatase activity, microsomes were prepared from cell lysates and mammary tissues by differential centrifugation using established methods (17). Briefly, tissue was sliced into small pieces and then ground in liquid nitrogen before being suspended in buffer (20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10% glycerol, 5 µM pepstatin and 5 µM leupeptin). The tissue lysate was then centrifuged for 20 min at 800 x g, and the pellet containing the nuclear fraction was discarded. The supernatant was subjected to ultracentrifugation (1 h at 100,000 x g) to separate microsomes from cytosol. To determine aromatase activity, microsomal protein was added to a 0.5-ml reaction mixture containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 5 mM glucose 6-phosphate, 5 units of glucose-6-phosphate dehydrogenase, 2 µM rotenone, and 1β-[3H]androstenedione (200 pM for microsomes for tissue, 12.5 nM for microsomes from cells). Following preincubation for 3 min, the reaction was initiated by the addition of 0.5 µM NADPH and allowed to run for up to several hours at 37 °C. Adding 3 ml of ice-cold chloroform, and applying vigorous shaking and brief centrifugation terminated the reaction. The resulting aqueous layer was further extracted with 3 ml of chloroform and treated with 0.5 ml of 5% activated charcoal/0.5% dextran. Following centrifugation of the mixture, the radioactivity in the supernatant was counted. Aromatase activity was quantified by measurement of the tritiated water released from 1β-[3H]androstenedione. The reaction was also performed in the presence of a specific aromatase inhibitor, as a specificity control and without NADPH as a background control. Aromatase activity was normalized to protein concentration.


Figure 5
View larger version (44K):
[in this window]
[in a new window]

  		FIGURE 5.
PGE2 via EP2 and EP4 induces expression of aromatase and suppresses levels of BRCA1 in adipocytes. Adipocytes were treated with the indicated concentration of PGE2 (A and B), butaprost (C), an EP2 agonist, or PGE1 alcohol (D), an EP4 agonist. E, cells were treated with vehicle (C), 500 nM PGE2, PGE2 plus EP2 antagonist (50 µM AH6809) or 500 nM PGE2 plus an EP4 antagonist (0.1 µM ONO AE3–208). All treatments were for 24 h. F, cells were first transfected with 2 µg of siRNAs to GFP, EP2 or EP4. 36 h after transfection, cells were treated with fresh medium containing vehicle (C) or 500 nM PGE2 for an additional 24 h. In A and C–F, total RNA was prepared and 10 µg/lane was subjected to Northern blotting. The blots were hybridized sequentially with the indicated probes. In B, cell lysates (100 µg of protein/lane) were analyzed by Western blotting for BRCA1 and β-actin, respectively.

 
Chromatin Immunoprecipitation Assay—ChIP assay was performed with a kit (Upstate) according to the manufacturer's instructions. 2 x 106 cells were cross-linked in a 1% formaldehyde solution for 10 min at 37 °C. Cells were then lysed in 200 µl of SDS buffer and sonicated to generate 200- to 1000-bp DNA fragments. After centrifugation, the cleared supernatant was diluted 10-fold with ChIP buffer and incubated with 1.5 µg of the indicated antibody at 4 °C. Immune complexes were precipitated, washed, and eluted as recommended. DNA-protein cross-links were reversed by heating at 65 °C for 4 h, and the DNA fragments were purified and dissolved in 50 µl of water. 10 µl of each sample was used as a template for PCR amplification. CYP19 oligonucleotide sequences for PCR primers were forward 5'-AAC CTG CTG ATG AAG TCA CAA-3'; and reverse, 5'-TCA GAC ATT TAG GCA AGA CT-3' (19). This primer set encompasses the CYP19 promoter I.3/II segment from nucleotide -302 to -38. PCR was performed at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s for 30 cycles. The PCR products generated from the ChIP template were sequenced, and the identity of the CYP19 promoter was confirmed. For real-time PCR analysis, ChIP-qPCR assay kits from Superarray Bioscience Corp. (Frederick, MD) were used. Real-time PCR was performed as described above.


Figure 6
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 6.
PGE2 via EP2 and EP4 induces aromatase and suppresses levels of BRCA1 in human breast cancer cells. A, SKBR3 cells were treated with the indicated concentration of PGE2, butaprost or PGE1 alcohol. B, cells were treated with vehicle, 500 nM PGE2, or 500 nM PGE2 plus 50 µM AH6809, an EP2 receptor antagonist, or 500 nM PGE2 plus 0.1 µM ONO AE3–208, an EP4 receptor antagonist. In A and B, aromatase activity was determined as under "Experimental Procedures." Enzyme activity is expressed as femtomoles/µg of protein/min. Means ± S.D. are shown, n = 6. *, p < 0.001. C and D, cells were treated with the indicated concentration of butaprost, an EP2 agonist, or PGE1 alcohol, an EP4 agonist. E, cells were treated with vehicle, 500 nM PGE2 or PGE2 plus the indicated concentration of AH6809. F, cells were treated with vehicle, 500 nM PGE2, or 500 nM PGE2 plus the indicated concentration of ONO AE3–208. All treatments were for 24 h. In C–F, total RNA was prepared and 10 µg/lane was subjected to Northern blotting. The blots were hybridized sequentially with the indicated probes.

 
Statistics—Comparisons between groups were made by Student's t test. A difference between groups of p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
EP2 and EP4 Are Important for PGE2-mediated Induction of Aromatase Expression—Initially, we showed that PGE2 caused dose-dependent induction of aromatase activity in human adipocytes (Fig. 1A). A pharmacological approach was then used to identify the EP receptors that mediated this inductive effect of PGE2. Similar to PGE2, butaprost, an EP2 agonist, caused a dose-dependent increase in aromatase activity and expression (Fig. 1, B–D). Next we evaluated AH6809, a nonselective EP2 receptor antagonist. This compound blocked PGE2-mediated induction of aromatase activity and expression (Fig. 1, E and F). Because both EP2 and EP4 signal via the cAMP->PKA->CREB pathway, we next evaluated the role of EP4 in mediating the induction of aromatase. Treatment with PGE1 alcohol, an EP4 agonist, caused dose-dependent induction of aromatase activity and expression (Fig. 2, A–C). ONO AE3–208, an EP4 antagonist, suppressed PGE2-mediated induction of aromatase activity and expression (Fig. 2, D and E). The inductive effects of the EP2 receptor agonist (butaprost) were blocked by the EP2 receptor antagonist (AH6809) but not by the EP4 receptor antagonist (ONO AE3–208) (Fig. 2F). Similarly, the EP4 antagonist blocked the inductive effects of PGE1 alcohol but not butaprost.

To complement this pharmacological approach, a genetic strategy was employed. As shown in Fig. 3A, siRNAs to EP2 and EP4 suppressed levels of EP2 and EP4, respectively. Consistent with the pharmacological findings, silencing of EP2 or EP4 blocked PGE2-mediated induction of aromatase expression (Fig. 3B) and activity (Fig. 3B). Although siRNAs to EP1 and EP3 suppressed levels of these EP receptors (Fig. 3A), we did not observe an effect on PGE2-mediated induction of aromatase expression and activity (Fig. 3B). Taken together, these data indicate that EP2 and EP4 mediate the inductive effects of PGE2 on aromatase expression in human adipocytes.

Signal Transduction Pathway by which EP2 and EP4 Regulate CYP19 Transcription—As mentioned above, both EP2 and EP4 can signal via the cAMP->PKA->CREB pathway. Accordingly, we investigated whether this pathway was involved in PGE2-mediated induction of aromatase. The induction of cAMP levels and PKA activity by PGE2 was suppressed by siRNAs to either EP2 or EP4 (Fig. 4, A and B). Moreover, siRNA to EP2 or EP4 blocked PGE2-mediated activation of the CYP19 promoter (Fig. 4C). ChIP assays were performed to evaluate the role of EP2 and EP4 in modulating the binding of p-CREB to the CYP19 promoter. Treatment with PGE2, an EP2 agonist (butaprost) or an EP4 agonist (PGE1 alcohol) stimulated the association between p-CREB and the CYP19 promoter (Fig. 4D, top panel). This interaction was blocked by pharmacological antagonists to EP2 (AH6809) or EP4 (ONO AE3–208) (Fig. 4D, middle panel) or siRNAs to these receptors (Fig. 4D, bottom panel).

Recently, PGE2 was found to suppress levels of BRCA1 resulting in enhanced CYP19 expression (19). Here we attempted to identify the EP receptors that are responsible for these effects. First, we confirmed that PGE2-mediated suppression of BRCA1 mRNA and protein levels occurred over a concentration range that also induced aromatase (Fig. 5, A and B, and supplemental Fig. S1A). Similar to PGE2, both the EP2 and EP4 receptor agonists suppressed BRCA1 levels and induced aromatase (Fig. 5, C and D; and supplemental Fig. S1, B and C). Consistent with these findings, the effects of PGE2 on BRCA1 and aromatase were blocked by antagonists to EP2 and EP4 (Fig. 5E and supplemental Fig. S1D) or silencing of these receptors (Fig. 5F and supplemental Fig. S1E).


Figure 7
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 7.
PGE2 via EP2 and EP4 enhanced p300 and suppressed BRCA1 recruitment to the CYP19 I. 3/II promoter in adipocytes. In A and C, adipocytes were transfected with 0.45 µg of CYP19 promoter and 0.2 µg of pSVβgal. In A, bars labeled BRCA1 represent cells that also received 0.45 µg of BRCA1 expression vector; bars labeled as BRCA1 mutant represent cells that also received 0.45 µg of mutant BRCA1 expression vector. In C, bars labeled p300 represent cells that received 0.45 µg of wild-type p300 expression vector, whereas bars labeled mutant p300 represent cells that received 0.45 µg of mutant p300 (lacks HAT activity) expression vector. In A and C, the total amount of DNA received by cells is limited to 2 µg by using vector DNA. 36 h after transfection, cells were treated with vehicle (control), 500 nM PGE2, EP2 agonist (1 µM butaprost) or EP4 agonist (0.2 µM PGE1 alcohol) for another 24 h. Luciferase activity was measured in cell lysates, and the activities represent data that have been normalized to β-galactosidase activity. Means ± S.D. are shown, n = 6. *, p < 0.01 compared with same treatment that received vector alone. B, ChIP assays were performed. In panels 1–4, cells were treated with vehicle (C), PGE2 (500 nM), butaprost (1 µM), or PGE1 alcohol (0.2 µM) for 2 h. Chromatin fragments were immunoprecipitated with antibodies against BRCA1 or p300, and the CYP19 I.3/II promoter was amplified by PCR (panels 1 and 3) or real-time PCR (panels 2 and 4). DNA sequencing was carried out, and the PCR product was confirmed to be the CYP19 1.3/II promoter. The CYP19 I.3/II promoter was not detected when normal IgG was used or antibody was omitted from immunoprecipitation step (data not shown). For panels 2 and 4, means ± S.D. are shown, n = 3. *, p < 0.01 versus control. D, cells were treated with vehicle or 500 nM PGE2 for 2 h. Cell lysates (500 µg) were subjected to immunoprecipitation with p300 antiserum and Western blotting was performed for BRCA1, p300 and phospho-CREB as indicated. These proteins were not immunoprecipitated with control IgG. Input is β-actin.

 
In breast cancer, increased aromatase activity occurs in tumor cells in addition to stromal cells (2). Hence, we also evaluated whether EP2 and EP4 were important for regulating aromatase expression and activity in a human breast cancer cell line (SKBR3 cells). Similar to the findings in adipocytes, treatment with PGE2, an EP2 receptor agonist (butaprost) or an EP4 receptor agonist (PGE1 alcohol) caused dose-dependent induction of aromatase activity and expression (Fig. 6, A, C and D, and supplemental Fig. S2, A and B). Both the EP2 (AH6809) and EP4 (ONO AE3–208) receptor antagonists suppressed PGE2-mediated induction of aromatase activity and expression (Fig. 6, B, E and F, and supplemental Fig. S2, C and D). Importantly, reciprocal changes in amounts of BRCA1 occurred (Fig. 6, C–F, and supplemental Fig. S2).

To further investigate the role of BRCA1 in repressing CYP19 transcription, transient transfections experiments were carried out. The induction of CYP19 promoter activity by PGE2 or agonists of EP2 and EP4 was suppressed by overexpressing wild-type BRCA1 but not a mutant form of BRCA1 that does not bind to DNA (Fig. 7A). ChIP assays were performed to further explore the interaction between BRCA1 and the CYP19 promoter. Treatment with PGE2 or agonists of EP2 and EP4 suppressed the interaction between BRCA1 and the CYP19 promoter (Fig. 7B). Because p300 is important for CREB-dependent activation of gene expression, the interaction between p300 and the CYP19 promoter was also investigated. Treatment with PGE2 or agonists of EP2 and EP4 increased the interaction between p300 and the CYP19 promoter (Fig. 7B). Hence, PGE2 via EP2 or EP4 reduced the interaction between BRCA1 and the CYP19 promoter but caused a reciprocal increase in the interaction between p300 and the CYP19 promoter. To further evaluate the role of p300, transient transfections were carried out. The induction of CYP19 promoter activity by PGE2 or agonists of EP2 and EP4 was suppressed by overexpressing a histone acetyltransferase mutant form of p300 (Fig. 7C). To further understand the transcriptional regulation of aromatase, we also investigated the interactions between BRCA1, p300, and p-CREB under basal conditions and following treatment with PGE2. In untreated cells, immunoprecipitation experiments suggested that BRCA1 and p300 were in a complex (Fig. 7D). Following treatment with PGE2, p300, and p-CREB were in the complex, but BRCA1 was not found.

In an effort to determine the in vivo relevance of these findings, an MMTV-COX-2 transgenic mouse model was employed. Previously, we utilized this model and demonstrated that overexpression of COX-2 led to both increased levels of intramammary PGE2 and aromatase activity (17). This was confirmed in the current study; overexpression of COX-2 in the mammary gland also led to increased aromatase activity and expression (Fig. 8). To evaluate whether the EP2 receptor contributed to PGE2-mediated induction of aromatase activity, MMTV-COX-2 EP2-/- mice were generated. Remarkably, when the EP2 receptor was absent, the COX-2-mediated increase in aromatase activity and expression was abrogated (Fig. 8). Consistent with our in vitro findings, levels of BRCA1 were also reduced in the mammary glands of MMTV-COX-2 transgenic mice, an effect that was reversed by knocking out EP2 (Fig. 8).

Because aromatase activity can be rate-limiting for the synthesis of estradiol, we also evaluated the role of EP2 and EP4 as determinants of estrogen-dependent gene expression. The progesterone receptor, an estrogen target gene, is positively regulated by an estrogen response element (31, 32). As shown in Fig. 9, treatment with PGE2 stimulated ERE-luciferase activity and induced the PR, effects that were suppressed by silencing of either EP2 or EP4.


Figure 8
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 8.
Increased levels of aromatase and reduced expression of BRCA1 are mediated by EP2 in the mammary glands of MMTV-COX-2 transgenic mice. Levels of aromatase and BRCA1 were determined in mammary glands from mice that varied in COX-2 (WT, wild-type; TG, transgenic) and EP2 (WT, wild-type; KO, knockout) status. In the top panel, aromatase activity was measured as described under "Experimental Procedures." Enzyme activity is expressed as femtomoles/mg of protein/h. Means ± S.D. are shown, n = 3; *, p < 0.01. In the bottom panel, total RNA was prepared from mammary glands from mice that varied in COX-2 and EP2 status. Northern blotting was performed (10 µg of RNA/lane), and the blots were sequentially hybridized with the indicated probes.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies indicate that COX-derived PGE2 can activate CYP19 transcription leading to enhanced aromatase activity and possibly an increased risk of breast cancer (9, 10, 17, 18, 33). Recent evidence suggests that the induction of aromatase by PGE2 is mediated, in part, by suppression of BRCA1, a repressor of CYP19 transcription (19). Given the established link between estrogen biosynthesis and the development and progression of hormone receptor-positive breast cancer, we have attempted to further elucidate the signaling pathway that mediates the activation of CYP19 transcription by PGE2. Several lines of evidence indicate that the EP2 and EP4 receptors are involved in mediating this inductive effect. First, agonists of EP2 and EP4 mimicked PGE2 in inducing aromatase expression. Moreover, EP2 and EP4 receptor antagonists blocked PGE2-mediated induction of aromatase. Consistent with these pharmacological findings, silencing of EP2 or EP4 suppressed PGE2-mediated induction of aromatase expression and activity. Although both EP2 and EP4 play a role in mediating the induction of aromatase, we note that inhibiting either receptor abrogated the stimulatory effects of PGE2. This finding suggests that EP2 and EP4 are functionally interdependent. There is growing evidence that G protein-coupled receptors can heterodimerize and that this physical interaction can affect the function of either receptor (34, 35). Recently, functionally important heterodimerization of the thromboxane and prostacyclin receptors (34) and the EP1 and β2-adrenergic receptors (35) was observed. Studies are underway to determine whether the current findings can be explained by heterodimerization of the EP2 and EP4 receptors.


Figure 9
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 9.
PGE2 via EP2 and EP4 induces estrogen receptor-dependent gene expression in adipocytes. A, adipocytes were first transfected with 2 µg of siRNAs to GFP, EP2, or EP4. 36 h later, fresh medium containing vehicle (C) or 500 nM PGE2 was added for an additional 24 h. In the panel on the left, cell lysates were analyzed by Western blotting for progesterone receptor (PR) protein levels using purified PR as a standard (Std). In the panel on the right, total RNA was isolated and PR mRNA was analyzed by real-time PCR. Values were normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase. Means ± S.D. are shown, n = 3, *, p < 0.01 versus PGE2-treated cells. B, cells were transfected with 0.9 µg of a ERE luciferase construct, 0.2 µg of pSVβgal, and 0.9 µg of siRNAs to GFP, EP2, or EP4. Thirty-six hours after transfection, cells were treated with vehicle (control) or 500 nM PGE2 for another 24 h. ERE luciferase activity represents data that have been normalized to β-galactosidase activity. Means ± S.D. are shown, n = 6, *, p < 0.01 compared with PGE2 treatment.

 
Because activation of EP2 or EP4 induced aromatase activity, we also investigated the role of these receptors in regulating estrogen-dependent gene expression. Silencing of EP2 or EP4 suppressed PGE2-mediated induction of the progesterone receptor, a prototypic estrogen-response gene. The significance of these in vitro findings is supported by evidence that the increase in aromatase expression and activity in the mammary glands of COX-2 transgenic mice was abrogated by knocking out EP2. Because the survival of EP4-deficient mice is poor, the role of EP4 as a determinant of aromatase expression was not assessed in vivo. A previous study suggested that EP1 was also a determinant of aromatase expression in adipose stromal cells (36). Although it's possible that EP1 signaling contributes to aromatase expression in this cell type, the earlier study did not utilize a genetic approach to confirm the results suggested by EP receptor agonists and antagonists. Our study was carried out in visceral adipocytes and a breast cancer cell line, because both cell types are believed to be relevant sources of estrogen in breast carcinogenesis. Whether our findings for EP2 and EP4 extend to other conditions, e.g. uterine leiomyomata, in which PGE2-mediated induction of aromatase appears to be important should be determined (37, 38).

Because EP2 and EP4 can signal via the cAMP->PKA->CREB pathway (23), we investigated whether this signaling pathway was responsible for PGE2-mediated activation of CYP19 transcription. The induction of cAMP levels and PKA activity by PGE2 was suppressed by silencing of EP2 or EP4. Furthermore, the PGE2-mediated increase in binding of p-CREB to the CYP19 I.3/II promoter was suppressed by EP2 and EP4 antagonists or silencing of these receptors. Although CYP19 transcription is regulated by CREB (11), little is known about the potential involvement of the coactivator CBP/p300 in regulating aromatase expression (39). We present evidence that PGE2 via EP2 and EP4 stimulated the interaction between p300 and the CYP19 I.3/II promoter. The interaction between p300 and pCREB was also enhanced by treatment with PGE2. The interaction between p300 and the CYP19 I.3/II promoter was functionally important, because overexpression of a p300 mutant that lacked histone acetyltransferase activity suppressed activation of the CYP19 I.3/II promoter by PGE2. Our observation that p300 is important in the regulation of CYP19 transcription provides mechanistic insights that may help to explain previous findings. More specifically, ligands of nuclear receptors, e.g. retinoids and PPAR-{gamma} ligands, have been reported to inhibit aromatase expression (40, 41). Ligands can stimulate an interaction between nuclear receptors and CBP/p300 (42, 43) that limits, in turn, the availability of relatively low concentrations of CBP/p300 to interact with transcription factors and enhance gene expression. Hence, the current results provide the basis for future experiments to determine whether squelching of CBP/p300 explains the ability of ligands of nuclear receptors to suppress the expression of aromatase.

The tumor suppressor BRCA1 plays a significant role in repressing aromatase expression (19, 44, 45). BRCA1 binds directly to the CYP19 I.3/II promoter region and suppresses transcription (19). Previously, PGE2 and other agents that stimulate cAMP signaling were found to suppress amounts of BRCA1 resulting in enhanced CYP19 transcription (19, 44). We extend upon these findings and show that PGE2-mediated suppression of BRCA1 is mediated by EP2 and EP4. In fact, EP2 and EP4 agonists mimicked PGE2 in suppressing levels of BRCA1 while inducing aromatase. Conversely, antagonists of EP2 and EP4 or silencing of these receptors blocked PGE2-mediated suppression of BRCA1. Additionally, PGE2 via EP2 and EP4 caused a decrease in the interaction between BRCA1 and the CYP19 I.3/II promoter. The reduced interaction between repressor and promoter was functionally important because overexpression of wild-type but not mutant BRCA1 abrogated PGE2-mediated activation of the aromatase promoter. Consistent with these in vitro findings, overexpression of COX-2 in the murine mammary gland, a known inducer of intramammary PGE2 (17), led to reduced levels of BRCA1 in association with increased aromatase expression; this effect was reversed by knocking out EP2. The mechanism by which PGE2 inhibits the expression of BRCA1 is likely to be complex. One recent study that utilized a human ovarian granulosa cell line demonstrated proteasome degradation of BRCA1 after a cAMP surge (46). Possibly, PGE2 via EP2 and EP4 acts, in part, by a similar mechanism to suppress levels of BRCA1 protein. Because PGE2 caused a reduction in BRCA1 mRNA in addition to BRCA1 protein, additional levels of regulation must also exist.

Regardless of the mechanism by which stimulation of EP2 or EP4 suppresses BRCA1 levels, it's important to consider the potential implications of this finding. In addition to inhibiting aromatase expression, BRCA1 is involved in several important cellular processes, including DNA damage control, DNA repair, chromatin remodeling, and mitotic spindle formation (47). BRCA1 is also involved in transcriptional regulation through interactions with a number of transcription factors (48). Hence, the reduction in amounts of BRCA1 mediated by activation of EP2 or EP4 is likely to affect multiple mechanisms that reduce the threshold for carcinogenesis. Taken together, PGE2 via EP2 and EP4 activates the cAMP->PKA->CREB pathway resulting in reciprocal changes in the interaction between BRCA1, p300, and the aromatase promoter I.3/II, which contributes, in turn, to enhanced CYP19 transcription and increased aromatase activity. Inhibitors of this pathway, including antagonists of EP2 and EP4, may reduce the risk of breast cancer.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant PO1CA77839, the Breast Cancer Research Foundation, the Botwinick-Wolfensohn Foundation (in memory of Mr. and Mrs. Benjamin Botwinick), and the Center for Cancer Prevention Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

1 To whom correspondence should be addressed: New York Presbyterian Hospital-Cornell, 525 East 68th St., Rm. F-203A, New York, NY 10065. Tel.: 212-746-4402; Fax: 212-746-4885; E-mail: ksubba{at}med.cornell.edu.

2 The abbreviations used are: PKA, protein kinase A; CREB, cAMP-response element-binding protein; COX, cyclooxygenase; PG, prostaglandin; PGE1, prostaglandin E1; PGE2, prostaglandin E2; PR, progesterone receptor; siRNA, small interference RNA; GFP, green fluorescent protein; ChIP, chromatin immunoprecipitation; ERE, estrogen response element. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., Graham-Lorence, S., Amarneh, B., Ito, Y., Fisher, C. R., Michael, M. D., Mendelson, C. R., and Bulun, S. E. (1994) Endocr. Rev. 15, 342-355[Abstract/Free Full Text]
  2. Miki, Y., Suzuki, T., Tazawa, C., Yamaguchi, Y., Kitada, K., Honma, S., Moriya, T., Hirakawa, H., Evans, D. B., Hayashi, S., Ohuchi, N., and Sasano, H. (2007) Cancer Res. 67, 3945-3954[Abstract/Free Full Text]
  3. Silva, M. C., Rowlands, M. G., Dowsett, M., Gusterson, B., McKinna, J. A., Fryatt, I., and Coombes, R. C. (1989) Cancer Res. 49, 2588-2591[Abstract/Free Full Text]
  4. Strassmer-Weippl, K., and Goss, P. E. (2003) J. Mammary Gland Biol. Neoplasia. 8, 5-18[CrossRef][Medline] [Order article via Infotrieve]
  5. Goss, P. E., Ingle, J. N., Martino, S., Robert, N. J., Muss, H. B., Piccart, M. J., Castiglione, M., Tu, D., Shepherd, L. E., Pritchard, K. I., Livingston, R. B., Davidson, N. E., Norton, L., Perez, E. A., Abrams, J. S., Therasse, P., Palmer, M. J., and Pater, J. L. (2003) N. Engl. J. Med. 349, 1793-1802[Abstract/Free Full Text]
  6. Mahendroo, M. S., Mendelson, C. R., and Simpson, E. R. (1993) J. Biol. Chem. 268, 19463-19470[Abstract/Free Full Text]
  7. Agarwal, V. R., Bulun, S. E., Leitch, M., Rohrich, R., and Simpson, E. R. (1996) J. Clin. Endocrinol. Metab. 81, 3843-3849[Abstract/Free Full Text]
  8. Chen, S., Itoh, T., Wu, K., Zhou, D., and Yang, C. (2002) J. Steroid. Biochem. Mol. Biol. 83, 93-99[CrossRef][Medline] [Order article via Infotrieve]
  9. Zhao, Y., Agarwal, V. R., Mendelson, C. R., and Simpson, E. R. (1996) Endocrinology 137, 5739-5742[Abstract]
  10. Zhao, Y., Agarwal, V. R., Mendelson, C. R., and Simpson, E. R. (1997) J. Steroid Biochem. Mol. Biol. 61, 203-210[Medline] [Order article via Infotrieve]
  11. Chen, S., Ye, J., Kijima, I., Kinoshita, Y., and Zhou, D. (2005) J. Steroid. Biochem. Mol. Biol. 95, 17-23[CrossRef][Medline] [Order article via Infotrieve]
  12. Prosperi, J. R., and Robertson, F. M. (2006) Prostaglandins Other Lipid Mediators 81, 55-70[CrossRef][Medline] [Order article via Infotrieve]
  13. Cai, Z., Kwintkiewicz, J., Young, M. E., and Stocco, C. (2007) Mol. Cell. Endocrinol. 263, 181-189[CrossRef][Medline] [Order article via Infotrieve]
  14. Brueggemeier, R. W., Quinn, A. L., Parrett, M. L., Joarder, F. S., Harris, R. E., and Robertson, F. M. (1999) Cancer Lett. 140, 27-35[CrossRef][Medline] [Order article via Infotrieve]
  15. Brodie, A. M., Lu, Q., Long, B. J., Fulton, A., Chen, T., Macpherson, N., DeJong, P. C., Blankenstein, M. A., Nortier, J. W. R., Slee, P. H. T. J., van de Ven, J., van Gorp, J. M. H. H., Elbers, J. R. J., Schipper, M. E. I., Blijham, G. H., and Thijssen, J. H. (2001) J. Steroid Biochem. Mol. Biol. 79, 41-47[CrossRef][Medline] [Order article via Infotrieve]
  16. Irahara, N., Miyoshi, Y., Taguchi, T., Tamaki, Y., and Noguchi, S. (2006) Int. J. Cancer 118, 1915-1921[CrossRef][Medline] [Order article via Infotrieve]
  17. Subbaramaiah, K., Howe, L. R., Port, E. R., Brogi, E., Fishman, J., Liu, C. H., Hla, T., Hudis, C., and Dannenberg, A. J. (2006) Cancer Res. 66, 5504-5511[Abstract/Free Full Text]
  18. Terry, M. B., Gammon, M. D., Zhang, F. F., Tawfik, H., Teitelbaum, S. L., Britton, J. A., Subbaramaiah, K., Dannenberg, A. J., and Neugut, A. I. (2004) JAMA 291, 2433-2440[Abstract/Free Full Text]
  19. Lu, M., Chen, D., Lin, Z., Reierstad, S., Trauernicht, A. M., Boyer, T. G., and Bulun, S. E. (2006) J. Clin. Endocrinol. Metab. 91, 4514-4519[Abstract/Free Full Text]
  20. Bresalier, R. S., Sandler, R. S., Quan, H., Bolognese, J. A., Oxenius, B., Horgan, K., Lines, C., Riddell, R., Morton, D., Lanas, A., Konstam, M. A., and Baron, J. A. (2005) N. Engl. J. Med. 352, 1092-1102[Abstract/Free Full Text]
  21. Solomon, S. D., McMurray, J. J. V., Pfeffer, M. A., Wittes, J., Fowler, R., Finn, P., Anderson, W. F., Zauber, A., Hawk, E., and Bertagnolli, M. (2005) N. Engl. J. Med. 352, 1071-1080[Abstract/Free Full Text]
  22. FitzGerald, G. A. (2004) N. Engl. J. Med. 351, 1709-1711[Free Full Text]
  23. Sugimoto, Y., and Narumiya, S. (2007) J. Biol. Chem. 282, 11613-11617[Abstract/Free Full Text]
  24. Dannenberg, A. J., and Subbaramaiah, K. (2003) Cancer Cell 4, 431-436[CrossRef][Medline] [Order article via Infotrieve]
  25. Hayes, F., Cayanan, C., Barilla, D., and Monteiro, A. N. A. (2000) Cancer Res. 60, 2411-2418[Abstract/Free Full Text]
  26. Zhou, D., and Chen, S. (1999) Arch. Biochem. Biophys. 371, 179-190[CrossRef][Medline] [Order article via Infotrieve]
  27. Chang, S. H., Ai, Y., Breyer, R. M., Lane, T. F., and Hla, T. (2005) Cancer Res. 65, 4496-4499[Abstract/Free Full Text]
  28. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  29. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
  30. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract/Free Full Text]
  31. Read, L. D., Snider, C. E., Miller, J. S., Greene, G. L., and Katzenellenbogen, B. S. (1988) Mol. Endocrinol. 2, 263-271[Abstract/Free Full Text]
  32. Savouret, J. F., Bailly, A., Misrahi, M., Rauch, C., Redeuilh, G., Chauchereau, A., and Milgrom, E. (1991) EMBO J. 10, 1875-1883[Medline] [Order article via Infotrieve]
  33. DuBois, R. N. (2004) JAMA 291, 2488-2489[Free Full Text]
  34. Wilson, S. J., Dowling, J. K., Zhao, L., Carnish, E., and Smyth, E. M. (2007) Arterioscler. Thromb. Vasc. Biol. 27, 290-296[Abstract/Free Full Text]
  35. Barnes, P. J. (2006) J. Clin. Invest. 116, 1210-1212[CrossRef][Medline] [Order article via Infotrieve]
  36. Richards, J. A., and Bruggemeier, R. W. (2003) J. Clin. Endocrinol. Metab. 88, 2810-2816[Abstract/Free Full Text]
  37. Noble, L. S., Takayama, K., Zeitoun, K. M., Putman, J. M., Johns, D. A., Hinshelwood, M. M., Agarwal, V. R., Zhao, Y., Carr, B. R., and Bulun, S. E. (1997) J. Clin. Endocrinol. Metab. 82, 600-606[Abstract/Free Full Text]
  38. Imir, A. G., Lin, Z., Yin, P., Deb, S., Yilmaz, B., Cetin, M., Cetin, A., and Bulun, S. E. (2007) J. Clin. Endocrinol. Metab. 92, 1979-1982[Abstract/Free Full Text]
  39. Bulun, S. E., Lin, Z., Imir, G., Amin, S., Demura, M., Yilmaz, B., Martin, R., Utsunomiya, H., Thung, S., Gurates, B., Tamura, M., Langoi, D., and Deb, S. (2005) Pharmacol. Rev. 57, 359-383[Abstract/Free Full Text]
  40. Rubin, G. L., Zhao, Y., Kalus, A. M., and Simpson, E. R. (2000) Cancer Res. 60, 1604-1608[Abstract/Free Full Text]
  41. Fan, W., Yanase, T., Morinaga, H., Mu, Y. M., Nomura, M., Okabe, T., Goto, K., Harada, N., and Nawata, H. (2005) Endocrinology 146, 85-92[CrossRef][Medline] [Order article via Infotrieve]
  42. Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S. C., Heyman, R. A., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1996) Cell 85, 403-414[CrossRef][Medline] [Order article via Infotrieve]
  43. Subbaramaiah, K., Cole, P. A., and Dannenberg, A. J. (2002) Cancer Res. 62, 2522-2530[Abstract/Free Full Text]
  44. Hu, Y., Ghosh, S., Amleh, A., Yue, W., Lu, Y., Katz, A., and Li, R. (2005) Oncogene 24, 8343-8348[CrossRef][Medline] [Order article via Infotrieve]
  45. Ghosh, S., Lu, Y., Katz, A., Hu, Y., and Li, R. (2007) Am. J. Physiol. Endocrinol. Metab. 292, E246-E252[Abstract/Free Full Text]
  46. Lu, Y., Amleh, A., Sun, J., Jin, X., McCullough, S. D., Baer, R., Ren, D., Li, R., and Hu, Y. (2007) Mol. Endocrinol. 21, 651-663[Abstract/Free Full Text]
  47. Starita, L. M., and Parvin, J. D. (2003) Curr. Opin. Cell Biol. 15, 345-350[CrossRef][Medline] [Order article via Infotrieve]
  48. Zhang, H., Somasundaram, K., Peng, Y., Tian, H., Zhang, H., Bi, D., Weber, B. L., and El-Deiry, W. S. (1998) Oncogene 16, 1713-1721[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
K. Subbaramaiah, R. Benezra, C. Hudis, and A. J. Dannenberg
Cyclooxygenase-2-derived Prostaglandin E2 Stimulates Id-1 Transcription
J. Biol. Chem., December 5, 2008; 283(49): 33955 - 33968.
[Abstract] [Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
D. W. Visscher, V. S. Pankratz, M. Santisteban, C. Reynolds, A. Ristimaki, R. A. Vierkant, W. L. Lingle, M. H. Frost, and L. C. Hartmann
Association Between Cyclooxygenase-2 Expression in Atypical Hyperplasia and Risk of Breast Cancer
J Natl Cancer Inst, March 19, 2008; 100(6): 421 - 427.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
283/6/3433    most recent
M705409200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Subbaramaiah, K.
Right arrow Articles by Dannenberg, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Subbaramaiah, K.
Right arrow Articles by Dannenberg, A. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement