Activation of Peroxisome Proliferator-Activated Receptor- and Retinoid X Receptor Inhibits Aromatase Transcription via Nuclear Factor-B
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内分泌学杂志 2005年第1期
Department of Medicine and Bioregulatory Science (W.F., T.Y., H.M., M.N., T.O., K.G., H.N.), Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 Japan; Core Research for Evolutional Science and Technology (CREST) (T.Y., H.M., M.N., T.O., K.G., H.N.), Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan; Department of Endocrinology (Y.-M.M.), Chinese PLA General Hospital, Beijing 100853, China; and Department of Biochemistry (N.H.), School of Medicine, Fujita Health University, 470-1192 Aichi, Japan
Address all correspondence and requests for reprints to: Toshihiko Yanase, M.D., Ph.D., Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: yanase@intmed3.med.kyushu-u.ac.jp.
Abstract
Our previous studies demonstrated that a peroxisome proliferator-activated receptor (PPAR)- ligand, troglitazone (TGZ),and/or a retinoid X receptor (RXR) ligand, LG100268 (LG), decreased the aromatase activity in both cultured human ovarian granulosa cells and human granulosa-like tumor KGN cells. In the present study, we further found that a combined treatment of TGZ+LG decreased aromatase promoter II (ArPII) activity in both ovarian KGN cells and fibroblast NIH-3T3 cells in a PPAR-dependent manner. Furthermore, the inhibition of both aromatase activity and the transcription of ArPII by TGZ+LG was completely eliminated when nuclear factor-B (NF-B) signaling was blocked by specific inhibitors, suggesting NF-B, which is endogenously expressed in both fibroblast and granulosa cells, might be a mediator of this inhibition. Interestingly, activation of NF-B by either forced expression of the p65 subunit or NF-B-inducing kinase up-regulated ArPII activity. Positive regulation of aromatase by endogenous NF-B was also suggested by the fact that NF-B-specific inhibitors suppress basal activity of the aromatase gene. A concomitant formation of high-order complex between NF-B p65 and ArPII was also observed by chromatin immunoprecipitation assay. Although activation of PPAR and RXR affected endogenous expression levels of neither inhibitory B nor p65, it impaired the interaction between NF-B and ArPII and the p65 based transcription as well. Altogether, these results indicate that activation of a nuclear receptor system, constituted by PPAR and RXR, down-regulates aromatase expression through the suppression of NF-B-dependent aromatase activation and thus provide a new insight in the mechanism of regulation of the aromatase gene.
Introduction
THE BIOSYNTHESIS OF estrogens is catalyzed by the enzyme complex referred to as aromatase cytochrome P-450, which aromatizes 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 (1). In humans, aromatase is present in many tissues, including ovary (2, 3), testis (4, 5), placenta (2), and brain (6, 7). The gene encoding the aromatase (CYP19) is extraordinarily long (more than 120 kb), with a coding region of approximately 30 kb, containing nine translated exons (II-X). One reason for this long gene is that the transcription of aromatase in different tissue is regulated by different promoters (8) (ovary: promoter II; placenta: promoter I.1; and adipose tissue: promoter I.4). The aromatase promoter II (ArPII) functions in the ovary under the control of FSH. In cooperation with Ad4BP/SF-1, FSH, via the cAMP-protein kinase A (PKA) pathway, stimulates aromatase gene expression in the ovary through promoter II.
It has been determined that estrogens contribute to the growth and development of some estrogen-dependent neoplasm, including breast, endometrial cancers, and some ovarian cancers (9, 10). Estrogens, especially those produced locally in the adipose stoma cells, exert a definite role in stimulating proliferation of breast tumor cells (11). In normal breast adipose tissue, the estrogen-producing aromatase gene is driven by a distal promoter I.4 (8), whereas in breast adipose tissue containing a tumor, there is a switch in the promoter, whereby the aromatase expression is regulated through the proximal promoter II. This shift results in elevated aromatase expression in the tumor or surrounding breast adipose tissue and subsequently elevated production of estrogen in local breast adipose tissue, thus leading to the development of breast cancer (12, 13, 14, 15, 16). These findings highlight the importance of promoter II, especially in breast cancer.
Peroxisome proliferator-activated receptor (PPAR)- is a nuclear receptor that has an essential role in adipogenesis and glucose homeostasis in response to its ligands, which are either naturally existing ligands like 15-deoxy-12,14 prostaglandin J2 or synthetic thiazolidinediones. Besides relatively well-known PPAR-expressing tissues like adipose tissue, adrenal gland, and spleen (17, 18, 19), ovary (20) and granulosa cells (21, 22) also express an abundant amount of PPAR, whose physiological role in these tissues is largely unknown. We previously reported that the PPAR ligand, troglitazone (TGZ), especially together with the retinoid X receptor (RXR) ligand, LG100268 (LG), dose-dependently inhibits aromatase activity in granulosa cells (21, 23, 24).
In the present study, we extended our study to clarify the underlying mechanism whereby activation of a nuclear receptor system constituted by PPAR and RXR down-regulates the aromatase gene. Herein we report an involvement of the transcriptional factor nuclear factor-B (NF-B) in the above mechanism as well as its importance in the regulation of aromatase expression through promoter II.
Materials and Methods
Materials
TGZ and LG were obtained from Sankyo Pharmaceuticals (Tokyo, Japan), and Ligand Pharmaceuticals Inc. (San Diego, CA), respectively. Caffeic acid phenethyl ester (CAPE), forskolin, and TNF were all purchased from Sigma-Aldrich (St. Louis, MO). Ammonium pyrrolidinedithiocarbamate (APDC) was purchased from Wako (Osaka, Japan). All the above compounds (except CAPE and TNF, which were dissolved in 50% ethanol and normal saline, respectively) were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of solvents (DMSO, 50% ethanol or normal saline) in the cell growth medium was 0.1% (vol/vol). An equal volume of solvents was added to control cultures during cell treatment with chemicals.
Cell culture
We established a human ovarian granulosa-like tumor cell line, KGN, from a 63-yr-old female patient with invasive granulosa cell carcinoma (25). The cells grew as an adherent monolayer with stable proliferation. The cells possess properties similar to those of normal granulosa cells, including the expression of functional FSH receptor and a relatively high aromatase activity, which is PKA dependent (25). The cells were maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) in an atmosphere of 5% CO2 at 37 C. NIH-3T3 cells were purchased from the Japanese Cell Research Bank (Tokyo) and maintained in DMEM (high glucose) supplemented with 10% FBS at 37 C.
Aromatase assay
The aromatase activity was determined by measuring the [3H]H2O released on conversion of [1?-3H]androstenedione to estrone, as described previously (21). The cells were precultured in 6-well plates in DMEM/F12 with 5% dextran-coated, charcoal-treated FBS for 48 h before treatment with chemicals. After the cells were treated with TGZ+LG, [1?-3H]androstenedione was added, and the cells were then further incubated for 6 h. In the case of combined treatment with the NF-B inhibitors, CAPE or APDC was added to cultures 2 h before an 8-h treatment with TGZ+LG. A second-round treatment consisting of 2 h of CAPE (or ADPC) followed by 8 h of TGZ+LG was carried out before addition of [1?-3H]androstenedione. Extraction of medium (2.0 ml) and measurement of radioactivity in [3H]H2O for aromatase activity were done as described previously (21). The amount of radioactivity was then standardized by protein concentration, which was determined using a micro-BCA kit (Pierce Chemical Co., Rockford, IL) and expressed as picomoles per milligram protein per 6 h.
Plasmid constructions
The 4.0-kb ArPII was amplified by PCR from genomic DNA. After confirmation of the entire sequence by direct sequencing, the fragment was subcloned into PGL3-Basic vector (Promega, Madison, WI) to make the luciferase reporter plasmid PGL3-ArPII, in which the luc+ gene is driven by the 4.0-kb fragment of human ArPII. To construct the NF-B luciferase reporter plasmid, pGL3-tk was first constructed by cloning the –109 to +37 region of the herpes virus thymidine kinase promoter into the BglII and HindIII sites of the pGL3-basic vector (Promega). A pair of oligonucleotides, 5'-TGGAAATTCCTGGAAATTCCTGGAAATTCC-3' and 5'-TCGAGGAATTTCCAGGAATTTCCAGGAATTTCCA-3', were annealed together, thus resulting in double-stranded oligonucleotides with both a blunt end and a XhoI compatible overhang, which were then ligated into the SmaI and XhoI sites of tk-Luc, thus giving rise to pGL3-NF-B containing three copies of the NF-B sites. The Renilla luciferase reporter plasmid phRL-cytomegalovirus (CMV), serving as an internal control in the dual-luciferase reporter assay, was purchased from Promega. Human p65 expression vector, pcDNA-p65, was provided by Dr. C. Scheidereit (Max Delbruck Center for Molecular Medicine, Berlin, Germany). pcDNA-NF-B-inducing kinase (NIK) was provided by Dr. D. Wallach (Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel). All plasmids were prepared from an overnight bacterial culture using the QIAfilter plasmid maxikit (Qiagen, Valencia, CA).
Relative luciferase reporter assay
For the relative luciferase reporter assay, 1.5 x 105 cells/well in 1 ml growth medium were seeded into 12-well plates, and 0.8 μg of PGL3-ArPII (or PGL3-NF-B) and 2.0 ng of phRL-CMV were transiently cotransfected in each well using the Superfect transfection reagent (Qiagen) following the manufacturer’s protocol. In the case of cotransfection, 0.15 μg of expression vector for p65 (pcDNA-p65) or NIK (pcDNA-NIK) was also added; the total amount of plasmid DNA added to each well was equalized using the empty vector: pcDNA-3.1. Twenty-four hours after transfection, the cells were treated with TGZ+LG for 24 h at the concentrations indicated in each figure. The cells were then lysed in 100 μl/well passive lysis buffer, and the luciferase assay was performed in accordance with the protocol of the dual-luciferase reporter assay system, using a Lumat LB 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany). The firefly luciferase activity, produced by PGL3-ArPII in identically treated triplicate samples, was normalized for the Renilla luciferase activity produced by phRL-CMV. The data shown are representative of at least three independent experiments. In the case of cotreatment with the NF-B inhibitors, cells were preincubated for 2 h with CAPE (working concentration 20 μg/ml) or APDC (working concentration 100 ng/ml) and then incubated for 10 h with TGZ+LG. Another round of 2 h of CAPE plus 10 h of TGZ+LG was carried out before the cells were lysed for luciferase assay.
Western blotting
NIH-3T3 and KGN cells treated with either TGZ+LG or DMSO were grown to subconfluent phase, washed with PBS, and actively lysed in 500 μl lysis buffer. Samples were subjected to electrophoresis on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were incubated with either a rabbit polyclonal antibody against the p65 subunit of NF-B [NF-B p65 (c-20): sc-372, Santa Cruz Biotechnology, Santa Cruz, CA] or a rabbit polyclonal antibody against inhibitory B (IB) (c-21: sc-371, Santa Cruz Biotechnology) and subsequently with a horseradish peroxidase-linked goat antirabbit IgG secondary antibody (Cell Signaling Technology, Beverly, MA). Detection was carried out using the ECL+Plus Western blotting detection system (Amersham Biosciences, Buckinghamshire, UK). Membranes were then visualized using a STORM 860 scanner (Molecular Dynamics, Sunnyvale, CA). Images were finally analyzed using ImageQuant software (Molecular Dynamics).
ChIP assays
These were performed by the chromatin immunoprecipitation (ChIP) assay kit (Upstate Biotechnology, Lake Placid, NY), according to the protocol provided by manufacturer with some modifications. Briefly, KGN cells were seeded in 10-cm2 dishes and treated overnight with 10 μM TGZ + 1 μM LG or the solvent DMSO. After an additional treatment of 10 ng/ml TNF or its solvent normal saline (NS) for 1 h, cells were cross-linked with 1% formaldehyde for 60 min, washed with chilled PBS, resuspended in 200 μl SDS lysis buffer, and sonicated six times for 10 sec each at 60% maximum setting of the sonicator (Handy Sonic-UR-20P, TOMY SEIKO Co., Ltd., Tokyo, Japan). Sonicated cell supernatant was diluted 10-fold, and 1% (20 μl) of the total diluted lysate was used for total genomic DNA as input DNA control. The rest (1980 μl) was then subjected to immunoclearing by 75 μl salmon sperm DNA/protein A agarose-50% slurry for 30 min at 4 C. Immunoprecipitation was performed for overnight at 4 C with 3 μg p65 antibody (Santa Cruz Biotechnology). For negative control, normal rabbit IgG (Santa Cruz Biotechnology) was used instead of p65 antibody. Precipitates were washed sequentially for 5 min each in low salt, high salt, LiCH immune complex wash buffers, and finally washed twice with Tris/EDTA buffer. Histone complexes were then eluted from the antibody by freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3). Histone-DNA cross-links (including the input samples) were reversed by 5 M NaCl at 65 C for 4 h. DNA fragments were extracted with a PCR purification kit (Qiagen). One microliter from a 30-μl DNA extraction was used for PCR and primed by sequences as follows: forward, 5'-GGG AAG AAG ATT GCC TAA AC-3'; reverse, 5'-TGT GGA AAT CAA AGG GAC AG-3'; the PCR size was 401 bp.
Real-time PCR
Immunoprecipitated DNA samples were then set to real-time PCR analysis to quantify the relative amount to their corresponding input controls with a LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instruction. Briefly, 1 μl immunoprecipitated DNA sample (or H2O as negative control), was placed into a 20-μl reaction volume containing 1 μl of each primer (10 μM) and 2 μl LightCycler-FastStart DNA Master SYBR Green I (Roche), which includes nucleotides, Tag DNA polymerase, and buffer. PCR products were visualized on a 2% agarose gel and finally validated by direct sequencing. Input samples were amplified simultaneously as the internal controls. Real-time PCR data for each immunoprecipitated sample were calculated as a ratio to its corresponding input sample. Briefly, threshold values (crossing line) obtained where fluorescent intensity was in the geometric phase, cycle number at the crossing point of an immunoprecipitated sample (Cip), and the corresponding input sample (Cco) were determined via LightCycler software version 3.5. The relative amount to input sample of the immunoprecipitated sample (Aip) was calculated by the formula of: Aip = 2(Cco–Cip).
Statistics
One-way ANOVA followed by Scheffé’s test was used for multigroup comparisons.
Results
TGZ+LG inhibited ArPII dose-dependently
We previously reported that TGZ+LG inhibit aromatase activity, and consistently the estrone production, in a dose-dependent manner (23). We also found that TGZ+LG down-regulated aromatase mRNA by both decreased transcription and increased degradation. Here a 4.0-kb fragment of human ArPII was inserted into PGL3-Basic to make the luciferase reporter PGL3-ArPII and then used to further address whether TGZ, LG, or TGZ+LG interfered with the transcription of aromatase from promoter II. KGN cells were transfected by Superfect with PGL3-ArPII as well as the internal control phRL-CMV. As shown in Fig. 1, on the addition of increasing concentration of TGZ+LG, the relative luciferase activity was decreased in a concentration-dependent manner. Although they were weaker, TGZ or LG alone also manifested inhibitory effects on ArPII. The results indicate that the inhibitory effect of TGZ+LG on aromatase gene is directly mediated by the inhibition of promoter II activity.
FIG. 1. TGZ+LG inhibits aromatase promoter II. The luciferase reporter PGL3-ArPII, wherein the luc+ gene is driven by a 4.0-kb segment of the human ArPII, was transfected into KGN cells, which were seeded in 12-well plates 1 d earlier. The cells were treated with an increasing concentration (as indicated) of TGZ, LG, or TGZ+LG 1 d after transfection for 24 h. Cells were then lysed and a dual-luciferase reporter assay was performed. The ArPII-mediated firefly luciferase signal was normalized using Renilla luciferase, which was constitutively expressed by the internal control phRL-CMV vector. Data expressed in mean ± SD was from identically treated triplicate samples of three independent experiments. , P < 0.05; *, P < 0.01, compared with basal level of the same treatment group.
PPAR is critical for the TGZ+LG inhibition
To further clarify the involvement of PPAR in the regulation of ArPII, the same experiment as described above was carried out in NIH-3T3 cells, which lack endogenous expression of PPAR (26). As shown in Fig. 2A, neither TGZ (or LG) alone nor combined treatment of TGZ+LG could decrease the expression of the PGL3-ArPII reporter, even when the concentration was raised to 10 μM for TGZ and 1.0 μM for LG, when the cells had been cotransfected with PGL3-ArPII+phRL-CMV and pcDNA3.1, the empty vector. However, on the exogenous cotransfection of the PPAR expression vector, either TGZ or LG alone significantly decreased ArPII activity in a concentration-dependent manner, and combined treatment of TGZ+LG caused a sharper decrease in expression from the promoter. This phenomenon nicely mimicked what was observed in KGN cells, which possess endogenous PPAR. These data clearly demonstrate the involvement of PPAR in the inhibition of ArPII.
FIG. 2. TGZ+LG inhibits ArPII in a PPAR-dependent manner. PPAR-deficient NIH-3T3 cells were transfected with PGL3-ArPII; either a human PPAR2 expression vector pcDNA3.1-PPAR2 or the empty control pcDNA3.1 vector was cotransfected. Cells were then treated with TGZ, LG, or both for 24 h. Neither TGZ (or LG) alone nor TGZ+LG inhibited the promoter in the absence of PPAR, whereas exogenous coexpression of the nuclear factor restored the inhibition. TGZ+LG synergistically inhibited the promoter in the presence of PPAR. , P < 0.05; *, P < 0.01, compared with basal level of the same treatment group.
NF-B inhibitors abolished the inhibition of TGZ+LG on aromatase gene
Due to the absence of a PPAR-RXR-responsive element in the aromatase promoter II (23), we previously suggested that PPAR might inhibit the promoter by an indirect mechanism (23). This hypothesis is supported by recent work (27), which showed that there was no binding of PPAR and RXR heterodimers to the promoter. A series of studies pointed out the inhibitory effect of PPAR activation on NF-B-dependent transcription system (28, 29, 30). We thus tested the possibility that PPAR activation inhibits aromatase gene through the NF-B system by using specific inhibitors for NF-B: CAPE (31) and APDC (32). CAPE specifically inhibits NF-B binding to DNA and also prevents the translocation of the p65 subunit of NF-B to the nucleus and delays IB resynthesis (31). As described above, cotransfection of PGL3-ArPII and pcDNA-PPAR in NIH-3T3 cells allowed direct assessment of PPAR mediation of the inhibitory effect on ArPII activity. CAPE was applied to this model to test the possible involvement of the NF-B system in the inhibition. As shown in Fig. 3A, NIH-3T3 cells were treated with 20 μg/ml CAPE or 50% ethanol for 2 h before combined treatment with TGZ+LG, which lasted for 8 h. To gain a clearer inhibition, a second round of 2 h of CAPE plus 8 h of TGZ+LG was carried out before the cells were subjected to luciferase assay. Pretreatment of the cells with CAPE completely abolished the inhibitory effect of PPAR activation, whereas the inhibition was still present with pretreatment with only the solvent for CAPE, 50% ethanol. A similar result was observed when CAPE was replaced by another NF-B inhibitor, APDC (data not shown).
FIG. 3. TGZ+LG inhibition disappeared on NF-B blockage. A, PGL3-ArPII was cotransfected with pcDNA3.1-PPAR2 into NIH-3T3 cells. Cells were first treated with or without 20 μg/ml CAPE for 2 h and then with TGZ+LG or the solvent DMSO for 8 h. A second round of 2 h of CAPE (or the solvent 50% ethanol) and 8 h of TGZ+LG (or DMSO) treatment was carried out before cells were lysed for the luciferase assay. CAPE abolished the inhibitory effect of PPAR activation and decreased the basal activity of the promoter as well. B, Aromatase activity assayed in KGN cells. Cells were treated with chemicals in the same manner as described in A. TGZ+LG inhibition of aromatase activity also disappeared on NF-B blockage. *, P < 0.01; NS, not significantly different.
We next assessed whether the inhibition of aromatase activity caused by TGZ+LG also disappears on treatment with CAPE.
KGN cells were treated with CAPE in the same manner as above before aromatase activities were assayed. As shown in Fig. 3B, on pretreatment with 50% ethanol, the solvent for CAPE, TGZ+LG significantly inhibited aromatase activity, whereas once the cells were pretreated with CAPE, no decrease of aromatase activity was seen. These results indicate the mediation of NF-B in the down-regulation of aromatase activity by TGZ+LG at the transcriptional level of promoter II.
NF-B up-regulates ArPII
In the experiments described in Fig. 3, we noticed that on treatment of CAPE, even basal levels of both ArPII and aromatase activity were decreased, suggesting that NF-B might be a positive regulator of aromatase gene. We tested this possibility by further experiments. As shown in Fig. 4A, cotransfection of the p65 subunit of NF-B directly stimulated ArPII by 4-fold in NIH-3T3 cells. A similar phenomenon was observed in KGN cells (data not shown). NIK, which causes degradation of IB by phosphorylation of the latter at serine 176 and thus activates p65 (33), was used to specifically induce the endogenous activation of NF-B. Figure 4B shows that PGL3-NF-B, (positive control, contains three repeats of the NF-B consensus elements) was augmented by NIK 2.80-fold. PGL-ArPII was also up-regulated 2.25 times by NIK. In the case of PGL3-Basic (negative control), NIK exhibited no effect.
FIG. 4. NF-B up-regulated ArPII. A, NIH-3T3 cells were transfected with PGL3-ArPII. pcDNA-p65 or pcDNA3.1 was cotransfected. ArPII activity was stimulated 4-fold by p65. B, pcDNA-NIK was transfected to NIH-3T3 cells to trigger the endogenous activation of NF-B, whose effect on ArPII was evaluated by cotransfection of PGL3-ArPII. PGL3-NF-B, a reporter containing three repeats of NF-B elements, and PGL3-basic was also included as positive and negative controls, respectively. The bars represent the relative effect of NIK on each reporter; namely, the NIK-mediated reporter activity was divided by the control pcDNA3.1 empty vector-mediated reporter activity. *, P < 0.01.
TGZ+LG interfered with the interaction between NF-B and ArPII
ChIP assay is a powerful technique to determine in vivo binding of transcription factors to target genes’ promoters on chromatin. We used this assay to evaluate the interaction of NF-B with ArPII, especially the recruitment of p65 to the promoter region. KGN cells pretreated either with an overnight 10 μM TGZ+1 μM LG or their solvent, DMSO, were challenged with 10 ng/ml TNF or its solvent NS for 1 h before being subjected to ChIP assay with an antibody against p65. Enrichment of ArPII DNA sequences in the chromatin immunoprecipitates, which indicates association of p65 to the promoter within intact chromatin, was visualized by PCR amplification. Based on our primitive promoter deletion analysis data, which show that a 600-bp ArPII reporter already responds positively to p65 and NIK, we designed the PCR to amplify a 401-bp region of ArPII (–403 to –2, upstream of ovary exon 2, GenBank accession no. D21241). As shown in Fig. 5A, although a weak band was amplified (30 cycles) in the absence of antibody (lane 1, which may represent the nonspecific binding of the ArPII chromatin region to normal rabbit IgG), an increase in the relative intensity of ArPII PCR band amplified from samples treated with p65 antibody indicated binding between the transcription factor and the promoter (lanes 2–5). Among cells not pretreated with TGZ+LG, 1 h TNF challenge seemed slightly increased band intensity (lane 3 vs. 2). Pretreatment of TGZ+LG clearly weakened the PCR band intensity (lane 4), suggesting a decreased occupancy by p65 on ArPII. However, the decrease was not observed in cells challenged with TNF (lane 5). Control amplification was with total input DNA (Fig 5A, lower panel). There was no change in the amplification of input DNA in all cases.
FIG. 5. ChIP assay of p65 binding to ArPII. KGN cells pretreated with or without an overnight 10.0 μM TGZ+1.0 μM LG were challenged with 10 ng/ml TNF or its solvent, NS, for 1 h. ChIP assay was then performed with anti-p65 antibody or normal rabbit IgG as negative control. Enrichment of ArPII-specific DNA sequence in immunoprecipitated DNA pool indicating association of p65 with ArPII within intact chromatin was visualized by PCR. A, PCR was performed on immunoprecipitated DNA pool with normal rabbit IgG [p65 Ab (–)], p65 Ab, and purified input DNA (input). Upper panel, PCR amplified ArPII-specific bands from cells under various treatments as indicated. Lower panel, ArPII PCR bands from input controls. B, Real-time PCR was performed to quantify the amount of immunoprecipitated ArPII DNA copy number from cells under various treatments relative to their corresponding input controls. *, P < 0.01; NS, not significantly different.
To further objectively tell the difference between different groups of cells, we performed real-time PCR to quantify the relative amount of immunoprecipitated ArPII copies to input control for each sample. Figure 5B shows that presence of p65 antibody significantly increased the relative copy number of immunoprecipitated ArPII DNA segments, indicating that p65 associates with ArPII. And TGZ+LG pretreatment significantly reduced the relative copy number, suggesting the association was impaired. However, TNF restored the TGZ+LG reduced relative ArPII copy number, although the cytokine did not change the copy number from cells not pretreated with TGZ+LG. Consistent with data presented in Fig. 4, these results indicated that NF-B may interact with ArPII in vivo, and activation of PPAR/RXR may interfere with the interaction.
The interference of PPAR activation on the endogenous expression of NF-B in KGN cells
The endogenous expression of the NF-B system in KGN cells was tested by Western blotting, using antibodies against the p65 subunit and IB, and was positively controlled using NIH3T3 cells, whose endogenous NF-B has already been proven (34). The KGN cells were treated with or without 24 h of 10 μM TGZ + 1.0 μM LG, actively lysed, and subjected to Western blotting. Figure 6 (upper panel) shows endogenous expression of IB in KGN cells and the lower panel the endogenous expression of the p65 subunit of NF-B. Neither of these two proteins’ expression was altered by a 24-h treatment of TGZ+LG, suggesting that PPAR activation does not interfere with NF-B function via down-regulation of p65 subunit expression or up-regulation of the IB protein.
FIG. 6. TGZ+LG did not alter the endogenous expression of IB and p65. KGN cells were treated with 10.0 μM TGZ+1.0 μM LG for 24 h and were then subjected to Western blotting analysis for the IB and p65 subunits of NF-B. NIH-3T3 cells were used as a positive control. KGN cells highly expressed both IB and p65, and 24 h of TGZ+LG did not apparently alter the protein expression levels.
PPAR activation suppresses NF-B transactivation
As shown above, PPAR activation does not apparently change the protein level of NF-B but impairs the interaction between the transcription factor and ArPII. We subsequently studied the possible interference of PPAR activation on NF-B transactivation in KGN cells. As shown in Fig. 7, in KGN cells, cotransfection of 0.15 μg/well of pcDNA-p65 stimulated PGL3-NF-B production (0.8 μg/well) approximately 4-fold, whereas the p65-augmented PGL3-NF-B signal was decreased in a concentration-dependent manner on cotreatment with an increasing concentration of TGZ+LG. Thus, activation of PPAR-RXR heterodimers by TGZ+LG resulted in inhibitory effects on NF-B-mediated transcription. Namely, the final net outcome effect of PPAR activation is a down-regulation of NF-B transactivation activity.
FIG. 7. TGZ+LG inhibited transactivation of NF-B. KGN cells were transfected with PGL3-NF-B, which contains three repeats of NF-B elements. A p65 expression vector (pcDNA-p65) or the pcDNA3.1 empty vector was also transfected. Cells cotransfected with p65 were further treated with increasing concentrations of TGZ+LG for 24 h. TGZ+LG was shown to decrease p65-stimulated PGL3-NF-B signal in a concentration-dependent pattern. *, P < 0.01.
Discussion
The physiological significance of the mysteriously high expression of PPAR in ovarian granulosa cells is largely unknown. We previously reported that the synthetic PPAR ligand, TGZ, in a concentration corresponding to human plasma TGZ concentration after oral administration of a therapeutic dosage, caused a significant decrease in aromatase activity as well as mRNA level in human ovarian granulosa cells (21). The effect was enhanced synergistically by the specific ligand (LG) for RXR, the PPAR partner. Consistently, TGZ+LG inhibited estrogen production in KGN cells (23), and TGZ reduced estrogen levels in patients with polycystic ovary syndrome, which suggested the in vivo relevance of the inhibition (35). In the present study, we further demonstrated that the aromatase promoter II, which is specially used in ovary, is also inhibited by TGZ+LG, indicating that inhibition occurs at the transcriptional level. It was recently reported that 15-deoxy-12,14 prostaglandin J2, which is believed to be the endogenous ligand for PPAR, inhibits aromatase activity through a PPAR-independent, but redox-sensitive, mechanism (36). However, TGZ and LG, the synthetic ligands for PPAR and RXR, respectively, seem to exert an inhibitory function in a PPAR-dependent way because the inhibition of ArPII could not be observed in PPAR-deficient NIH-3T3 cells unless the nuclear receptor is exogenously expressed. It is noteworthy that even for LG-induced inhibition, PPAR was required, suggesting that inhibition requires PPAR-RXR heterodimers. The important involvement of PPAR in the regulation of the aromatase gene was also strengthened by a recent report that demonstrated that an environmental toxin, a commonly used plasticizer, di-C2-ethylhexyl phthalate, decreased aromatase expression through both PPAR and PPAR in granulosa cells (37).
The aromatase gene is unique in that expression of the gene in different tissues is driven by different promoters in a tissue-specific pattern (8). Promoter II is typically used to drive the gene in ovarian granulosa cells, especially before menopause. Local estrogen production in breast adipose tissue has a definite mitogenic role in breast tumors (15, 16), and local estrogen levels in breast tumors were found 10 times higher than that in the circulation of postmenopausal women (38). Although in normal adipose tissue aromatase is mainly produced via the promoter I.4 (15, 39), the local accumulation of estrogen in breast adipose tissue containing a tumor is largely due to a critical shift in promoter usage from I.4 to II (12, 13, 14, 40). In this study, we found that TGZ+LG, in a PPAR-dependent manner, dose-dependently inhibited ArPII activity in ovarian KGN cells as well as in fibroblast NIH-3T3 cells, suggesting that the PPAR inhibitory effect on ArPII might be universal. These data highlighted the importance of ArPII and the therapeutic potency of TGZ+LG.
Due to the lack of an apparent consensus about PPAR-responsive element on ArPII, we hypothesized that the inhibition mechanism might be indirect (23). This idea is supported by a recent study, which shows that PPAR is unable to bind ArPII (27).
It has been proven that the ArPII gains its maximal activity when both PKA and protein kinase C are activated by cotreatment with forskolin and tetradecanoyl phorbol acetate (41, 42). NF-B is one of the transcriptional factors that can be activated by the activation of the protein kinase C pathway (34). Whereas on the other hand, activation of PPAR can regulate inflammatory responses by suppressing the activation of the transcriptional factor of NF-B (28, 29, 30). In the present study, we tested the hypothesis that PPAR activation may exert its inhibitory effect on ArPII by inhibiting NF-B, which is endogenously expressed in ovarian granulosa cells and breast tissues as well (43). In this study the inhibitory effect of PPAR-RXR activation on both ArPII and aromatase activity was found to sharply disappear on treatment with NF-B blockers (either CAPE or APDC), suggesting that NF-B might be the mediator of this inhibition. If this is the case, NF-B should logically be a positive regulator of ArPII. In line with this, the basal ArPII activity as well as the aromatase activity was decreased on CAPE treatment, and activation of the NF-B system by either forced expression of p65 or cotransfection of NIK to activate endogenous NF-B stimulated ArPII activity. Consistently, ChIP assay also showed the interaction between NF-B and ArPII. However, no classical consensus NF-B-responsive element was detected on the promoter. Nevertheless, because there is an instance that NF-B may bind a DNA motif, which is not related to the classical NF-B consensus sequence (44), we suppose that there might exist putative ArPII-specific binding sites for p65, which are to be further delineated.
Although we observed no effect of TGZ+LG on endogenous expressions of either IB or p65, which is considered one possible mechanism by which NF-B system is regulated (45). Treatment of TGZ+LG apparently weakened the interaction between p65 and ArPII, suggesting activation of PPAR may interfere with the formation of high-order complex between NF-B and aromatase gene at chromatin level. This is probably further explained by the finding that activated PPAR can physically interact with p65 and results in inhibition of NF-B (46, 47). And probably as an outcome of the impaired transcription factor-promoter association, we found that PPAR activation by TGZ+LG suppressed the transactivation ability of NF-B. The suppression by the PPAR-RXR nuclear receptor system may also possibly be related to the fact that the nuclear receptors compete for limited amounts of the general coactivators, cAMP response element-binding protein and steroid receptor coactivator-1, as we previously reported (48).
Considering our previous finding that activation of a PPAR-RXR nuclear receptor system by TGZ+LG inhibits aromatase by accelerating mRNA degradation, we report in the present study that TGZ+LG inhibited transcriptional activity of the ArPII in a PPAR-dependent manner. These data reinforce the potential use of synthetic PPAR and RXR agonists for therapeutic applications in diseases in which estrogens, locally or systematically, play prominent pathogenic roles, especially in diseases like breast cancer. In addition, we found that the inhibition disappeared on blockage of NF-B, which was found in turn to positively regulate aromatase. Notably, activation of NF-B has been found involved in the proliferation and metastasis of breast cancer cells (43), for which, although several mechanisms have been suggested, we suppose that stimulation of aromatase might be an additional one. Classically, regulation of ArPII involves PKA-cAMP response element-binding protein (49) and the orphan nuclear receptor steroidogenic factor 1 (Ad4BP/SF-1) (50), but the actual regulation may be much more complicated, at least in that nuclear receptors like PPAR, RXR, and their cross-talk with the transcriptional factor NF-B might also play some important roles.
References
Thompson Jr EA, Siiteri PK 1974 Utilization of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatization of androstenedione. J Biol Chem 249:5364–5372
Means GD, Kilgore MW, Mahendroo MS, Mendelson CR, Simpson ER 1991 Tissue-specific promoters regulate aromatase cytochrome P450 gene expression in human ovary and fetal tissues. Mol Endocrinol 5:2005–2013
Jenkins C, Michael D, Mahendroo M, Simpson E 1993 Exon-specific northern analysis and rapid amplification of cDNA ends (RACE) reveal that the proximal promoter II (PII) is responsible for aromatase cytochrome P450 (CYP19) expression in human ovary. Mol Cell Endocrinol 97:R1–R6
Tsai-Morris CH, Aquilana DR, Dufau ML 1985 Cellular localization of rat testicular aromatase activity during development. Endocrinology 116:38–46
Nitta H, Bunick D, Hess RA, Janulis L, Newton SC, Millette CF, Osawa Y, Shizuta Y, Toda K, Bahr JM 1993 Germ cells of the mouse testis express aromatase. Endocrinology 132:1396–1401
Naftolin F, Ryan KJ, Davies IJ, Reddy VV, Flores F, Petro Z, Kuhn M, White RJ, Takaoka Y, Wolin L 1975 The formation of estrogens by central neuroendocrine tissues. Recent Prog Horm Res 31:295–319
Roselli CE, Horton LE, Resko JA 1985 Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system. Endocrinology 117:2471–2477
Simpson ER, Michael MD, Agarwal VR, Hinshelwood MM, Bulun SE, Zhao Y 1997 Cytochromes P450 11: expression of the CYP19 (aromatase) gene: an unusual case of alternative promoter usage. FASEB J 11:29–36
Bokhman JV 1983 Two pathogenetic types of endometrial carcinoma. Gynecol Oncol 15:10–17
Sasano H, Harada N 1998 Intratumoral aromatase in human breast, endometrial, and ovarian malignancies. Endocr Rev 19:593–607
Yue W, Wang JP, Hamilton CJ, Demers LM, Santen RJ 1998 In situ aromatization enhances breast tumor estradiol levels and cellular proliferation. Cancer Res 58:927–932
Agarwal VR, Bulun SE, Leitch M, Rohrich R, Simpson ER 1996 Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients. J Clin Endocrinol Metab 81:3843–3849
Zhou C, Zhou D, Esteban J, Murai J, Siiteri PK, Wilczynski S, Chen S 1996 Aromatase gene expression and its exon I usage in human breast tumors. J Steroid Biochem Mol Biol 59:163–171
Harada N 1997 Aberrant expression of aromatase in breast cancer tissues. J Steroid Biochem Mol Biol 61:175–184
Bulun SE, Simpson ER 1994 Competitive reverse transcription-polymerase chain reaction analysis indicates that levels of aromatase cytochrome P450 transcripts in adipose tissue of buttocks, thighs, and abdomen of women increase with advancing age. J Clin Endocrinol Metab 78:428–432
Bulun SE, Price TM, Aitken J, Mahendroo MS, Simpson ER 1993 A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription. J Clin Endocrinol Metab 77:1622–1628
Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ, Umesono K, Evans RM 1994 Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc Natl Acad Sci USA 91:7355–7359
Lemberger T, Desvergne B, Wahli W 1996 PPARs: a nuclear receptor-signaling pathway in lipid metabolism. Annu Rev Cell Dev Biol 12:335–363
Tontonoz P, Hu E, Graves RA, Budavari AI, Spiegelman BM 1994 mPPAR 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev 8:1224–1234
Braissant O, Foufelle F, Scotto C, Dauca M, Wahli W 1996 Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-, -?, and - in the adult rat. Endocrinology 137:354–366
Mu YM, Yanase T, Nishi Y, Waseda N, Oda T, Tanaka A, Takayanagi R, Nawata H 2000 Insulin sensitizer, troglitazone, directly inhibits aromatase activity in human ovarian granulosa cells. Biochem Biophys Res Commun 271:710–713
Komar CM, Braissant O, Wahli W, Curry Jr TE 2001 Expression and localization of PPARs in the rat ovary during follicular development and the periovulatory period. Endocrinology 142:4831–4838
Mu YM, Yanase T, Nishi Y, Takayanagi R, Goto K, Nawata H 2001 Combined treatment with specific ligands for PPAR:RXR nuclear receptor system markedly inhibits the expression of cytochrome aromatase in human granulosa cancer cells. Mol Cell Endocrinol 181:239–248
Yanase T, Mu YM, Nishi Y, Goto K, Nomura M, Okabe T, Takayanagi R, Nawata H 2001 Regulation of aromatase by nuclear receptors. J Steroid Biochem Mol Biol 79:187–192
Nishi Y, Yanase T, Mu Y, Oba K, Ichino I, Saito M, Nomura M, Mukasa C, Okabe T, Goto K, Takayanagi R, Kashimura Y, Haji M, Nawata H 2001 Establishment and characterization of a steroidogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor. Endocrinology 142:437–445
Tontonoz P, Hu E, Spiegelman BM 1994 Stimulation of adipogenesis in fibroblasts by PPAR 2, a lipid-activated transcription factor. Cell 79:1147–1156
Rubin GL, Duong JH, Clyne CD, Speed CJ, Murata Y, Gong C, Simpson ER 2003 Ligands for the peroxisomal proliferator-activated receptor and the retinoid X receptor inhibit aromatase cytochrome P450 (CYP19) expression mediated by promoter II in human breast adipose. Endocrinology 143:2863–2871
Wang N, Verna L, Chen NG, Chen J, Li H, Forman BM, Stemerman MB 2002 Constitutive activation of peroxisome proliferator-activated receptor suppresses pro-inflammatory adhesion molecules in human vascular endothelial cells. J Biol Chem 277:34176–34181
Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK 1998 The peroxisome proliferator-activated receptor- is a negative regulator of macrophage activation. Nature 391:79–82
Jiang C, Ting AT, Seed B 1998 PPAR- agonists inhibit production of monocyte inflammatory cytokines. Nature 391:82–86
Natarajan K, Singh S, Burke Jr TR, Grunberger D, Aggarwal BB 1996 Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-B. Proc Natl Acad Sci USA 93:9090–9095
Fujii A, Harada T, Yamauchi N, Iwabe T, Nishi Y, Yanase T, Nawata H, Terakawa N 2003 Interleukin-8 gene and protein expression are up-regulated by interleukin-1? in normal human ovarian cells and a granulosa tumor cell line. Fertil Steril 79:151–157
Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB 1999 NF-B activation by tumor necrosis factor requires the Akt serine-threonine kinase. Nature 401:82–85
Diaz-Meco MT, Berra E, Municio MM, Sanz L, Lozano J, Dominguez I, Diaz-Golpe V, Lain de Lera MT, Alcami J, Paya CV 1993 A dominant negative protein kinase C subspecies blocks NF-B activation. Mol Cell Biol 13:4770–4775
Dunaif A, Scott D, Finegood D, Quintana B, Whitcomb R 1996 The insulin-sensitizing agent troglitazone improves metabolic and reproductive abnormalities in the polycystic ovary syndrome. J Clin Endocrinol Metab 81:3299–3306
Winnett G, van Hagen D, Schrey M 2003 Prostaglandin J2 metabolites inhibit aromatase activity by redox-sensitive mechanisms: potential implications for breast cancer therapy. Int J Cancer 103:600–605
Lovekamp-Swan T, Jetten AM, Davis BJ 2003 Dual activation of PPAR and PPAR by mono-(2-ethylhexyl) phthalate in rat ovarian granulosa cells. Mol Cell Endocrinol 201:133–141
Van Landeghem AA, Poortman J, Nabuurs M, Thijssen JH 1985 Endogenous concentration and subcellular distribution of estrogens in normal and malignant human breast tissue. Cancer Res 45:2900–2906
Grodin JM, Siiteri PK, MacDonald PC 1973 Source of estrogen production in postmenopausal women. J Clin Endocrinol Metab 36:207–214
Bulun SE, Mahendroo MS, Simpson ER 1994 Aromatase gene expression in adipose tissue: relationship to breast cancer. J Steroid Biochem Mol Biol 49:319–326
Zhao Y, Agarwal VR, Mendelson CR, Simpson ER 1996 Estrogen biosynthesis proximal to a breast tumor is stimulated by PGE2 via cyclic AMP, leading to activation of promoter II of the CYP19 (aromatase) gene. Endocrinology 137:5739–5742
Evans CT, Corbin CJ, Saunders CT, Merill JC, Simpson ER, Mendelson CR 1987 Regulation of estrogen biosynthesis in human adipose stromal cells. Effects of dibutyryl cyclic AMP, epidermal growth factor, and phorbol esters on the synthesis of aromatase cytochrome P-450. J Biol Chem 262:6914–6920
Watabe M, Hishikawa K, Takayanagi A, Shimizu N, Nakaki T 2004 Caffeic acid phenethyl ester induces apoptosis by inhibition of NFB and activation of Fas in human breast cancer MCF-7 cells. J Biol Chem 279:6017–6026
Todorov VT, Volkl S, Muller M, Bohla A, Klar J, Kunz-Schughart LA, Hehlgans T, Kurtz A 2004 Tumor necrosis factor- activates NFB to inhibit renin transcription by targeting cAMP-responsive element. J Biol Chem 279:1458–1467
Delerive P, Gervois P, Fruchart JC, Staels B 2000 Induction of IB expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor- activators. J Biol Chem 275:36703–36707
Chen F, Wang M, O’Connor JP, He M, Tripathi T, Harrison LE 2003 Phosphorylation of PPAR via active ERK1/2 leads to its physical association with p65 and inhibition of NF-?. J Cell Biochem 90:732–744
Chung SW, Kang BY, Kim SH, Pak YK, Cho D, Trinchieri G, Kim TS 2000 Oxidized low density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptor- and nuclear factor-B. J Biol Chem 275:32681–32687
Uchimura K, Nakamuta M, Enjoji M, Irie T, Sugimoto R, Muta T, Iwamoto H, Nawata H 2001 Activation of retinoic X receptor and peroxisome proliferator-activated receptor- inhibits nitric oxide and tumor necrosis factor- production in rat Kupffer cells. Hepatology 33:91–99
Michael MD, Michael LF, Simpson ER 1997 A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 (CYP19) gene. Mol Cell Endocrinol 134:147–156
Michael MD, Kilgore MW, Morohashi K, Simpson ER 1995 Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary. J Biol Chem 270:13561–13566(WuQiang Fan, Toshihiko Ya)
Address all correspondence and requests for reprints to: Toshihiko Yanase, M.D., Ph.D., Department of Medicine and Bioregulatory Science, Graduate School of Medical Science, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: yanase@intmed3.med.kyushu-u.ac.jp.
Abstract
Our previous studies demonstrated that a peroxisome proliferator-activated receptor (PPAR)- ligand, troglitazone (TGZ),and/or a retinoid X receptor (RXR) ligand, LG100268 (LG), decreased the aromatase activity in both cultured human ovarian granulosa cells and human granulosa-like tumor KGN cells. In the present study, we further found that a combined treatment of TGZ+LG decreased aromatase promoter II (ArPII) activity in both ovarian KGN cells and fibroblast NIH-3T3 cells in a PPAR-dependent manner. Furthermore, the inhibition of both aromatase activity and the transcription of ArPII by TGZ+LG was completely eliminated when nuclear factor-B (NF-B) signaling was blocked by specific inhibitors, suggesting NF-B, which is endogenously expressed in both fibroblast and granulosa cells, might be a mediator of this inhibition. Interestingly, activation of NF-B by either forced expression of the p65 subunit or NF-B-inducing kinase up-regulated ArPII activity. Positive regulation of aromatase by endogenous NF-B was also suggested by the fact that NF-B-specific inhibitors suppress basal activity of the aromatase gene. A concomitant formation of high-order complex between NF-B p65 and ArPII was also observed by chromatin immunoprecipitation assay. Although activation of PPAR and RXR affected endogenous expression levels of neither inhibitory B nor p65, it impaired the interaction between NF-B and ArPII and the p65 based transcription as well. Altogether, these results indicate that activation of a nuclear receptor system, constituted by PPAR and RXR, down-regulates aromatase expression through the suppression of NF-B-dependent aromatase activation and thus provide a new insight in the mechanism of regulation of the aromatase gene.
Introduction
THE BIOSYNTHESIS OF estrogens is catalyzed by the enzyme complex referred to as aromatase cytochrome P-450, which aromatizes 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 (1). In humans, aromatase is present in many tissues, including ovary (2, 3), testis (4, 5), placenta (2), and brain (6, 7). The gene encoding the aromatase (CYP19) is extraordinarily long (more than 120 kb), with a coding region of approximately 30 kb, containing nine translated exons (II-X). One reason for this long gene is that the transcription of aromatase in different tissue is regulated by different promoters (8) (ovary: promoter II; placenta: promoter I.1; and adipose tissue: promoter I.4). The aromatase promoter II (ArPII) functions in the ovary under the control of FSH. In cooperation with Ad4BP/SF-1, FSH, via the cAMP-protein kinase A (PKA) pathway, stimulates aromatase gene expression in the ovary through promoter II.
It has been determined that estrogens contribute to the growth and development of some estrogen-dependent neoplasm, including breast, endometrial cancers, and some ovarian cancers (9, 10). Estrogens, especially those produced locally in the adipose stoma cells, exert a definite role in stimulating proliferation of breast tumor cells (11). In normal breast adipose tissue, the estrogen-producing aromatase gene is driven by a distal promoter I.4 (8), whereas in breast adipose tissue containing a tumor, there is a switch in the promoter, whereby the aromatase expression is regulated through the proximal promoter II. This shift results in elevated aromatase expression in the tumor or surrounding breast adipose tissue and subsequently elevated production of estrogen in local breast adipose tissue, thus leading to the development of breast cancer (12, 13, 14, 15, 16). These findings highlight the importance of promoter II, especially in breast cancer.
Peroxisome proliferator-activated receptor (PPAR)- is a nuclear receptor that has an essential role in adipogenesis and glucose homeostasis in response to its ligands, which are either naturally existing ligands like 15-deoxy-12,14 prostaglandin J2 or synthetic thiazolidinediones. Besides relatively well-known PPAR-expressing tissues like adipose tissue, adrenal gland, and spleen (17, 18, 19), ovary (20) and granulosa cells (21, 22) also express an abundant amount of PPAR, whose physiological role in these tissues is largely unknown. We previously reported that the PPAR ligand, troglitazone (TGZ), especially together with the retinoid X receptor (RXR) ligand, LG100268 (LG), dose-dependently inhibits aromatase activity in granulosa cells (21, 23, 24).
In the present study, we extended our study to clarify the underlying mechanism whereby activation of a nuclear receptor system constituted by PPAR and RXR down-regulates the aromatase gene. Herein we report an involvement of the transcriptional factor nuclear factor-B (NF-B) in the above mechanism as well as its importance in the regulation of aromatase expression through promoter II.
Materials and Methods
Materials
TGZ and LG were obtained from Sankyo Pharmaceuticals (Tokyo, Japan), and Ligand Pharmaceuticals Inc. (San Diego, CA), respectively. Caffeic acid phenethyl ester (CAPE), forskolin, and TNF were all purchased from Sigma-Aldrich (St. Louis, MO). Ammonium pyrrolidinedithiocarbamate (APDC) was purchased from Wako (Osaka, Japan). All the above compounds (except CAPE and TNF, which were dissolved in 50% ethanol and normal saline, respectively) were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of solvents (DMSO, 50% ethanol or normal saline) in the cell growth medium was 0.1% (vol/vol). An equal volume of solvents was added to control cultures during cell treatment with chemicals.
Cell culture
We established a human ovarian granulosa-like tumor cell line, KGN, from a 63-yr-old female patient with invasive granulosa cell carcinoma (25). The cells grew as an adherent monolayer with stable proliferation. The cells possess properties similar to those of normal granulosa cells, including the expression of functional FSH receptor and a relatively high aromatase activity, which is PKA dependent (25). The cells were maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) in an atmosphere of 5% CO2 at 37 C. NIH-3T3 cells were purchased from the Japanese Cell Research Bank (Tokyo) and maintained in DMEM (high glucose) supplemented with 10% FBS at 37 C.
Aromatase assay
The aromatase activity was determined by measuring the [3H]H2O released on conversion of [1?-3H]androstenedione to estrone, as described previously (21). The cells were precultured in 6-well plates in DMEM/F12 with 5% dextran-coated, charcoal-treated FBS for 48 h before treatment with chemicals. After the cells were treated with TGZ+LG, [1?-3H]androstenedione was added, and the cells were then further incubated for 6 h. In the case of combined treatment with the NF-B inhibitors, CAPE or APDC was added to cultures 2 h before an 8-h treatment with TGZ+LG. A second-round treatment consisting of 2 h of CAPE (or ADPC) followed by 8 h of TGZ+LG was carried out before addition of [1?-3H]androstenedione. Extraction of medium (2.0 ml) and measurement of radioactivity in [3H]H2O for aromatase activity were done as described previously (21). The amount of radioactivity was then standardized by protein concentration, which was determined using a micro-BCA kit (Pierce Chemical Co., Rockford, IL) and expressed as picomoles per milligram protein per 6 h.
Plasmid constructions
The 4.0-kb ArPII was amplified by PCR from genomic DNA. After confirmation of the entire sequence by direct sequencing, the fragment was subcloned into PGL3-Basic vector (Promega, Madison, WI) to make the luciferase reporter plasmid PGL3-ArPII, in which the luc+ gene is driven by the 4.0-kb fragment of human ArPII. To construct the NF-B luciferase reporter plasmid, pGL3-tk was first constructed by cloning the –109 to +37 region of the herpes virus thymidine kinase promoter into the BglII and HindIII sites of the pGL3-basic vector (Promega). A pair of oligonucleotides, 5'-TGGAAATTCCTGGAAATTCCTGGAAATTCC-3' and 5'-TCGAGGAATTTCCAGGAATTTCCAGGAATTTCCA-3', were annealed together, thus resulting in double-stranded oligonucleotides with both a blunt end and a XhoI compatible overhang, which were then ligated into the SmaI and XhoI sites of tk-Luc, thus giving rise to pGL3-NF-B containing three copies of the NF-B sites. The Renilla luciferase reporter plasmid phRL-cytomegalovirus (CMV), serving as an internal control in the dual-luciferase reporter assay, was purchased from Promega. Human p65 expression vector, pcDNA-p65, was provided by Dr. C. Scheidereit (Max Delbruck Center for Molecular Medicine, Berlin, Germany). pcDNA-NF-B-inducing kinase (NIK) was provided by Dr. D. Wallach (Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel). All plasmids were prepared from an overnight bacterial culture using the QIAfilter plasmid maxikit (Qiagen, Valencia, CA).
Relative luciferase reporter assay
For the relative luciferase reporter assay, 1.5 x 105 cells/well in 1 ml growth medium were seeded into 12-well plates, and 0.8 μg of PGL3-ArPII (or PGL3-NF-B) and 2.0 ng of phRL-CMV were transiently cotransfected in each well using the Superfect transfection reagent (Qiagen) following the manufacturer’s protocol. In the case of cotransfection, 0.15 μg of expression vector for p65 (pcDNA-p65) or NIK (pcDNA-NIK) was also added; the total amount of plasmid DNA added to each well was equalized using the empty vector: pcDNA-3.1. Twenty-four hours after transfection, the cells were treated with TGZ+LG for 24 h at the concentrations indicated in each figure. The cells were then lysed in 100 μl/well passive lysis buffer, and the luciferase assay was performed in accordance with the protocol of the dual-luciferase reporter assay system, using a Lumat LB 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany). The firefly luciferase activity, produced by PGL3-ArPII in identically treated triplicate samples, was normalized for the Renilla luciferase activity produced by phRL-CMV. The data shown are representative of at least three independent experiments. In the case of cotreatment with the NF-B inhibitors, cells were preincubated for 2 h with CAPE (working concentration 20 μg/ml) or APDC (working concentration 100 ng/ml) and then incubated for 10 h with TGZ+LG. Another round of 2 h of CAPE plus 10 h of TGZ+LG was carried out before the cells were lysed for luciferase assay.
Western blotting
NIH-3T3 and KGN cells treated with either TGZ+LG or DMSO were grown to subconfluent phase, washed with PBS, and actively lysed in 500 μl lysis buffer. Samples were subjected to electrophoresis on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were incubated with either a rabbit polyclonal antibody against the p65 subunit of NF-B [NF-B p65 (c-20): sc-372, Santa Cruz Biotechnology, Santa Cruz, CA] or a rabbit polyclonal antibody against inhibitory B (IB) (c-21: sc-371, Santa Cruz Biotechnology) and subsequently with a horseradish peroxidase-linked goat antirabbit IgG secondary antibody (Cell Signaling Technology, Beverly, MA). Detection was carried out using the ECL+Plus Western blotting detection system (Amersham Biosciences, Buckinghamshire, UK). Membranes were then visualized using a STORM 860 scanner (Molecular Dynamics, Sunnyvale, CA). Images were finally analyzed using ImageQuant software (Molecular Dynamics).
ChIP assays
These were performed by the chromatin immunoprecipitation (ChIP) assay kit (Upstate Biotechnology, Lake Placid, NY), according to the protocol provided by manufacturer with some modifications. Briefly, KGN cells were seeded in 10-cm2 dishes and treated overnight with 10 μM TGZ + 1 μM LG or the solvent DMSO. After an additional treatment of 10 ng/ml TNF or its solvent normal saline (NS) for 1 h, cells were cross-linked with 1% formaldehyde for 60 min, washed with chilled PBS, resuspended in 200 μl SDS lysis buffer, and sonicated six times for 10 sec each at 60% maximum setting of the sonicator (Handy Sonic-UR-20P, TOMY SEIKO Co., Ltd., Tokyo, Japan). Sonicated cell supernatant was diluted 10-fold, and 1% (20 μl) of the total diluted lysate was used for total genomic DNA as input DNA control. The rest (1980 μl) was then subjected to immunoclearing by 75 μl salmon sperm DNA/protein A agarose-50% slurry for 30 min at 4 C. Immunoprecipitation was performed for overnight at 4 C with 3 μg p65 antibody (Santa Cruz Biotechnology). For negative control, normal rabbit IgG (Santa Cruz Biotechnology) was used instead of p65 antibody. Precipitates were washed sequentially for 5 min each in low salt, high salt, LiCH immune complex wash buffers, and finally washed twice with Tris/EDTA buffer. Histone complexes were then eluted from the antibody by freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3). Histone-DNA cross-links (including the input samples) were reversed by 5 M NaCl at 65 C for 4 h. DNA fragments were extracted with a PCR purification kit (Qiagen). One microliter from a 30-μl DNA extraction was used for PCR and primed by sequences as follows: forward, 5'-GGG AAG AAG ATT GCC TAA AC-3'; reverse, 5'-TGT GGA AAT CAA AGG GAC AG-3'; the PCR size was 401 bp.
Real-time PCR
Immunoprecipitated DNA samples were then set to real-time PCR analysis to quantify the relative amount to their corresponding input controls with a LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instruction. Briefly, 1 μl immunoprecipitated DNA sample (or H2O as negative control), was placed into a 20-μl reaction volume containing 1 μl of each primer (10 μM) and 2 μl LightCycler-FastStart DNA Master SYBR Green I (Roche), which includes nucleotides, Tag DNA polymerase, and buffer. PCR products were visualized on a 2% agarose gel and finally validated by direct sequencing. Input samples were amplified simultaneously as the internal controls. Real-time PCR data for each immunoprecipitated sample were calculated as a ratio to its corresponding input sample. Briefly, threshold values (crossing line) obtained where fluorescent intensity was in the geometric phase, cycle number at the crossing point of an immunoprecipitated sample (Cip), and the corresponding input sample (Cco) were determined via LightCycler software version 3.5. The relative amount to input sample of the immunoprecipitated sample (Aip) was calculated by the formula of: Aip = 2(Cco–Cip).
Statistics
One-way ANOVA followed by Scheffé’s test was used for multigroup comparisons.
Results
TGZ+LG inhibited ArPII dose-dependently
We previously reported that TGZ+LG inhibit aromatase activity, and consistently the estrone production, in a dose-dependent manner (23). We also found that TGZ+LG down-regulated aromatase mRNA by both decreased transcription and increased degradation. Here a 4.0-kb fragment of human ArPII was inserted into PGL3-Basic to make the luciferase reporter PGL3-ArPII and then used to further address whether TGZ, LG, or TGZ+LG interfered with the transcription of aromatase from promoter II. KGN cells were transfected by Superfect with PGL3-ArPII as well as the internal control phRL-CMV. As shown in Fig. 1, on the addition of increasing concentration of TGZ+LG, the relative luciferase activity was decreased in a concentration-dependent manner. Although they were weaker, TGZ or LG alone also manifested inhibitory effects on ArPII. The results indicate that the inhibitory effect of TGZ+LG on aromatase gene is directly mediated by the inhibition of promoter II activity.
FIG. 1. TGZ+LG inhibits aromatase promoter II. The luciferase reporter PGL3-ArPII, wherein the luc+ gene is driven by a 4.0-kb segment of the human ArPII, was transfected into KGN cells, which were seeded in 12-well plates 1 d earlier. The cells were treated with an increasing concentration (as indicated) of TGZ, LG, or TGZ+LG 1 d after transfection for 24 h. Cells were then lysed and a dual-luciferase reporter assay was performed. The ArPII-mediated firefly luciferase signal was normalized using Renilla luciferase, which was constitutively expressed by the internal control phRL-CMV vector. Data expressed in mean ± SD was from identically treated triplicate samples of three independent experiments. , P < 0.05; *, P < 0.01, compared with basal level of the same treatment group.
PPAR is critical for the TGZ+LG inhibition
To further clarify the involvement of PPAR in the regulation of ArPII, the same experiment as described above was carried out in NIH-3T3 cells, which lack endogenous expression of PPAR (26). As shown in Fig. 2A, neither TGZ (or LG) alone nor combined treatment of TGZ+LG could decrease the expression of the PGL3-ArPII reporter, even when the concentration was raised to 10 μM for TGZ and 1.0 μM for LG, when the cells had been cotransfected with PGL3-ArPII+phRL-CMV and pcDNA3.1, the empty vector. However, on the exogenous cotransfection of the PPAR expression vector, either TGZ or LG alone significantly decreased ArPII activity in a concentration-dependent manner, and combined treatment of TGZ+LG caused a sharper decrease in expression from the promoter. This phenomenon nicely mimicked what was observed in KGN cells, which possess endogenous PPAR. These data clearly demonstrate the involvement of PPAR in the inhibition of ArPII.
FIG. 2. TGZ+LG inhibits ArPII in a PPAR-dependent manner. PPAR-deficient NIH-3T3 cells were transfected with PGL3-ArPII; either a human PPAR2 expression vector pcDNA3.1-PPAR2 or the empty control pcDNA3.1 vector was cotransfected. Cells were then treated with TGZ, LG, or both for 24 h. Neither TGZ (or LG) alone nor TGZ+LG inhibited the promoter in the absence of PPAR, whereas exogenous coexpression of the nuclear factor restored the inhibition. TGZ+LG synergistically inhibited the promoter in the presence of PPAR. , P < 0.05; *, P < 0.01, compared with basal level of the same treatment group.
NF-B inhibitors abolished the inhibition of TGZ+LG on aromatase gene
Due to the absence of a PPAR-RXR-responsive element in the aromatase promoter II (23), we previously suggested that PPAR might inhibit the promoter by an indirect mechanism (23). This hypothesis is supported by recent work (27), which showed that there was no binding of PPAR and RXR heterodimers to the promoter. A series of studies pointed out the inhibitory effect of PPAR activation on NF-B-dependent transcription system (28, 29, 30). We thus tested the possibility that PPAR activation inhibits aromatase gene through the NF-B system by using specific inhibitors for NF-B: CAPE (31) and APDC (32). CAPE specifically inhibits NF-B binding to DNA and also prevents the translocation of the p65 subunit of NF-B to the nucleus and delays IB resynthesis (31). As described above, cotransfection of PGL3-ArPII and pcDNA-PPAR in NIH-3T3 cells allowed direct assessment of PPAR mediation of the inhibitory effect on ArPII activity. CAPE was applied to this model to test the possible involvement of the NF-B system in the inhibition. As shown in Fig. 3A, NIH-3T3 cells were treated with 20 μg/ml CAPE or 50% ethanol for 2 h before combined treatment with TGZ+LG, which lasted for 8 h. To gain a clearer inhibition, a second round of 2 h of CAPE plus 8 h of TGZ+LG was carried out before the cells were subjected to luciferase assay. Pretreatment of the cells with CAPE completely abolished the inhibitory effect of PPAR activation, whereas the inhibition was still present with pretreatment with only the solvent for CAPE, 50% ethanol. A similar result was observed when CAPE was replaced by another NF-B inhibitor, APDC (data not shown).
FIG. 3. TGZ+LG inhibition disappeared on NF-B blockage. A, PGL3-ArPII was cotransfected with pcDNA3.1-PPAR2 into NIH-3T3 cells. Cells were first treated with or without 20 μg/ml CAPE for 2 h and then with TGZ+LG or the solvent DMSO for 8 h. A second round of 2 h of CAPE (or the solvent 50% ethanol) and 8 h of TGZ+LG (or DMSO) treatment was carried out before cells were lysed for the luciferase assay. CAPE abolished the inhibitory effect of PPAR activation and decreased the basal activity of the promoter as well. B, Aromatase activity assayed in KGN cells. Cells were treated with chemicals in the same manner as described in A. TGZ+LG inhibition of aromatase activity also disappeared on NF-B blockage. *, P < 0.01; NS, not significantly different.
We next assessed whether the inhibition of aromatase activity caused by TGZ+LG also disappears on treatment with CAPE.
KGN cells were treated with CAPE in the same manner as above before aromatase activities were assayed. As shown in Fig. 3B, on pretreatment with 50% ethanol, the solvent for CAPE, TGZ+LG significantly inhibited aromatase activity, whereas once the cells were pretreated with CAPE, no decrease of aromatase activity was seen. These results indicate the mediation of NF-B in the down-regulation of aromatase activity by TGZ+LG at the transcriptional level of promoter II.
NF-B up-regulates ArPII
In the experiments described in Fig. 3, we noticed that on treatment of CAPE, even basal levels of both ArPII and aromatase activity were decreased, suggesting that NF-B might be a positive regulator of aromatase gene. We tested this possibility by further experiments. As shown in Fig. 4A, cotransfection of the p65 subunit of NF-B directly stimulated ArPII by 4-fold in NIH-3T3 cells. A similar phenomenon was observed in KGN cells (data not shown). NIK, which causes degradation of IB by phosphorylation of the latter at serine 176 and thus activates p65 (33), was used to specifically induce the endogenous activation of NF-B. Figure 4B shows that PGL3-NF-B, (positive control, contains three repeats of the NF-B consensus elements) was augmented by NIK 2.80-fold. PGL-ArPII was also up-regulated 2.25 times by NIK. In the case of PGL3-Basic (negative control), NIK exhibited no effect.
FIG. 4. NF-B up-regulated ArPII. A, NIH-3T3 cells were transfected with PGL3-ArPII. pcDNA-p65 or pcDNA3.1 was cotransfected. ArPII activity was stimulated 4-fold by p65. B, pcDNA-NIK was transfected to NIH-3T3 cells to trigger the endogenous activation of NF-B, whose effect on ArPII was evaluated by cotransfection of PGL3-ArPII. PGL3-NF-B, a reporter containing three repeats of NF-B elements, and PGL3-basic was also included as positive and negative controls, respectively. The bars represent the relative effect of NIK on each reporter; namely, the NIK-mediated reporter activity was divided by the control pcDNA3.1 empty vector-mediated reporter activity. *, P < 0.01.
TGZ+LG interfered with the interaction between NF-B and ArPII
ChIP assay is a powerful technique to determine in vivo binding of transcription factors to target genes’ promoters on chromatin. We used this assay to evaluate the interaction of NF-B with ArPII, especially the recruitment of p65 to the promoter region. KGN cells pretreated either with an overnight 10 μM TGZ+1 μM LG or their solvent, DMSO, were challenged with 10 ng/ml TNF or its solvent NS for 1 h before being subjected to ChIP assay with an antibody against p65. Enrichment of ArPII DNA sequences in the chromatin immunoprecipitates, which indicates association of p65 to the promoter within intact chromatin, was visualized by PCR amplification. Based on our primitive promoter deletion analysis data, which show that a 600-bp ArPII reporter already responds positively to p65 and NIK, we designed the PCR to amplify a 401-bp region of ArPII (–403 to –2, upstream of ovary exon 2, GenBank accession no. D21241). As shown in Fig. 5A, although a weak band was amplified (30 cycles) in the absence of antibody (lane 1, which may represent the nonspecific binding of the ArPII chromatin region to normal rabbit IgG), an increase in the relative intensity of ArPII PCR band amplified from samples treated with p65 antibody indicated binding between the transcription factor and the promoter (lanes 2–5). Among cells not pretreated with TGZ+LG, 1 h TNF challenge seemed slightly increased band intensity (lane 3 vs. 2). Pretreatment of TGZ+LG clearly weakened the PCR band intensity (lane 4), suggesting a decreased occupancy by p65 on ArPII. However, the decrease was not observed in cells challenged with TNF (lane 5). Control amplification was with total input DNA (Fig 5A, lower panel). There was no change in the amplification of input DNA in all cases.
FIG. 5. ChIP assay of p65 binding to ArPII. KGN cells pretreated with or without an overnight 10.0 μM TGZ+1.0 μM LG were challenged with 10 ng/ml TNF or its solvent, NS, for 1 h. ChIP assay was then performed with anti-p65 antibody or normal rabbit IgG as negative control. Enrichment of ArPII-specific DNA sequence in immunoprecipitated DNA pool indicating association of p65 with ArPII within intact chromatin was visualized by PCR. A, PCR was performed on immunoprecipitated DNA pool with normal rabbit IgG [p65 Ab (–)], p65 Ab, and purified input DNA (input). Upper panel, PCR amplified ArPII-specific bands from cells under various treatments as indicated. Lower panel, ArPII PCR bands from input controls. B, Real-time PCR was performed to quantify the amount of immunoprecipitated ArPII DNA copy number from cells under various treatments relative to their corresponding input controls. *, P < 0.01; NS, not significantly different.
To further objectively tell the difference between different groups of cells, we performed real-time PCR to quantify the relative amount of immunoprecipitated ArPII copies to input control for each sample. Figure 5B shows that presence of p65 antibody significantly increased the relative copy number of immunoprecipitated ArPII DNA segments, indicating that p65 associates with ArPII. And TGZ+LG pretreatment significantly reduced the relative copy number, suggesting the association was impaired. However, TNF restored the TGZ+LG reduced relative ArPII copy number, although the cytokine did not change the copy number from cells not pretreated with TGZ+LG. Consistent with data presented in Fig. 4, these results indicated that NF-B may interact with ArPII in vivo, and activation of PPAR/RXR may interfere with the interaction.
The interference of PPAR activation on the endogenous expression of NF-B in KGN cells
The endogenous expression of the NF-B system in KGN cells was tested by Western blotting, using antibodies against the p65 subunit and IB, and was positively controlled using NIH3T3 cells, whose endogenous NF-B has already been proven (34). The KGN cells were treated with or without 24 h of 10 μM TGZ + 1.0 μM LG, actively lysed, and subjected to Western blotting. Figure 6 (upper panel) shows endogenous expression of IB in KGN cells and the lower panel the endogenous expression of the p65 subunit of NF-B. Neither of these two proteins’ expression was altered by a 24-h treatment of TGZ+LG, suggesting that PPAR activation does not interfere with NF-B function via down-regulation of p65 subunit expression or up-regulation of the IB protein.
FIG. 6. TGZ+LG did not alter the endogenous expression of IB and p65. KGN cells were treated with 10.0 μM TGZ+1.0 μM LG for 24 h and were then subjected to Western blotting analysis for the IB and p65 subunits of NF-B. NIH-3T3 cells were used as a positive control. KGN cells highly expressed both IB and p65, and 24 h of TGZ+LG did not apparently alter the protein expression levels.
PPAR activation suppresses NF-B transactivation
As shown above, PPAR activation does not apparently change the protein level of NF-B but impairs the interaction between the transcription factor and ArPII. We subsequently studied the possible interference of PPAR activation on NF-B transactivation in KGN cells. As shown in Fig. 7, in KGN cells, cotransfection of 0.15 μg/well of pcDNA-p65 stimulated PGL3-NF-B production (0.8 μg/well) approximately 4-fold, whereas the p65-augmented PGL3-NF-B signal was decreased in a concentration-dependent manner on cotreatment with an increasing concentration of TGZ+LG. Thus, activation of PPAR-RXR heterodimers by TGZ+LG resulted in inhibitory effects on NF-B-mediated transcription. Namely, the final net outcome effect of PPAR activation is a down-regulation of NF-B transactivation activity.
FIG. 7. TGZ+LG inhibited transactivation of NF-B. KGN cells were transfected with PGL3-NF-B, which contains three repeats of NF-B elements. A p65 expression vector (pcDNA-p65) or the pcDNA3.1 empty vector was also transfected. Cells cotransfected with p65 were further treated with increasing concentrations of TGZ+LG for 24 h. TGZ+LG was shown to decrease p65-stimulated PGL3-NF-B signal in a concentration-dependent pattern. *, P < 0.01.
Discussion
The physiological significance of the mysteriously high expression of PPAR in ovarian granulosa cells is largely unknown. We previously reported that the synthetic PPAR ligand, TGZ, in a concentration corresponding to human plasma TGZ concentration after oral administration of a therapeutic dosage, caused a significant decrease in aromatase activity as well as mRNA level in human ovarian granulosa cells (21). The effect was enhanced synergistically by the specific ligand (LG) for RXR, the PPAR partner. Consistently, TGZ+LG inhibited estrogen production in KGN cells (23), and TGZ reduced estrogen levels in patients with polycystic ovary syndrome, which suggested the in vivo relevance of the inhibition (35). In the present study, we further demonstrated that the aromatase promoter II, which is specially used in ovary, is also inhibited by TGZ+LG, indicating that inhibition occurs at the transcriptional level. It was recently reported that 15-deoxy-12,14 prostaglandin J2, which is believed to be the endogenous ligand for PPAR, inhibits aromatase activity through a PPAR-independent, but redox-sensitive, mechanism (36). However, TGZ and LG, the synthetic ligands for PPAR and RXR, respectively, seem to exert an inhibitory function in a PPAR-dependent way because the inhibition of ArPII could not be observed in PPAR-deficient NIH-3T3 cells unless the nuclear receptor is exogenously expressed. It is noteworthy that even for LG-induced inhibition, PPAR was required, suggesting that inhibition requires PPAR-RXR heterodimers. The important involvement of PPAR in the regulation of the aromatase gene was also strengthened by a recent report that demonstrated that an environmental toxin, a commonly used plasticizer, di-C2-ethylhexyl phthalate, decreased aromatase expression through both PPAR and PPAR in granulosa cells (37).
The aromatase gene is unique in that expression of the gene in different tissues is driven by different promoters in a tissue-specific pattern (8). Promoter II is typically used to drive the gene in ovarian granulosa cells, especially before menopause. Local estrogen production in breast adipose tissue has a definite mitogenic role in breast tumors (15, 16), and local estrogen levels in breast tumors were found 10 times higher than that in the circulation of postmenopausal women (38). Although in normal adipose tissue aromatase is mainly produced via the promoter I.4 (15, 39), the local accumulation of estrogen in breast adipose tissue containing a tumor is largely due to a critical shift in promoter usage from I.4 to II (12, 13, 14, 40). In this study, we found that TGZ+LG, in a PPAR-dependent manner, dose-dependently inhibited ArPII activity in ovarian KGN cells as well as in fibroblast NIH-3T3 cells, suggesting that the PPAR inhibitory effect on ArPII might be universal. These data highlighted the importance of ArPII and the therapeutic potency of TGZ+LG.
Due to the lack of an apparent consensus about PPAR-responsive element on ArPII, we hypothesized that the inhibition mechanism might be indirect (23). This idea is supported by a recent study, which shows that PPAR is unable to bind ArPII (27).
It has been proven that the ArPII gains its maximal activity when both PKA and protein kinase C are activated by cotreatment with forskolin and tetradecanoyl phorbol acetate (41, 42). NF-B is one of the transcriptional factors that can be activated by the activation of the protein kinase C pathway (34). Whereas on the other hand, activation of PPAR can regulate inflammatory responses by suppressing the activation of the transcriptional factor of NF-B (28, 29, 30). In the present study, we tested the hypothesis that PPAR activation may exert its inhibitory effect on ArPII by inhibiting NF-B, which is endogenously expressed in ovarian granulosa cells and breast tissues as well (43). In this study the inhibitory effect of PPAR-RXR activation on both ArPII and aromatase activity was found to sharply disappear on treatment with NF-B blockers (either CAPE or APDC), suggesting that NF-B might be the mediator of this inhibition. If this is the case, NF-B should logically be a positive regulator of ArPII. In line with this, the basal ArPII activity as well as the aromatase activity was decreased on CAPE treatment, and activation of the NF-B system by either forced expression of p65 or cotransfection of NIK to activate endogenous NF-B stimulated ArPII activity. Consistently, ChIP assay also showed the interaction between NF-B and ArPII. However, no classical consensus NF-B-responsive element was detected on the promoter. Nevertheless, because there is an instance that NF-B may bind a DNA motif, which is not related to the classical NF-B consensus sequence (44), we suppose that there might exist putative ArPII-specific binding sites for p65, which are to be further delineated.
Although we observed no effect of TGZ+LG on endogenous expressions of either IB or p65, which is considered one possible mechanism by which NF-B system is regulated (45). Treatment of TGZ+LG apparently weakened the interaction between p65 and ArPII, suggesting activation of PPAR may interfere with the formation of high-order complex between NF-B and aromatase gene at chromatin level. This is probably further explained by the finding that activated PPAR can physically interact with p65 and results in inhibition of NF-B (46, 47). And probably as an outcome of the impaired transcription factor-promoter association, we found that PPAR activation by TGZ+LG suppressed the transactivation ability of NF-B. The suppression by the PPAR-RXR nuclear receptor system may also possibly be related to the fact that the nuclear receptors compete for limited amounts of the general coactivators, cAMP response element-binding protein and steroid receptor coactivator-1, as we previously reported (48).
Considering our previous finding that activation of a PPAR-RXR nuclear receptor system by TGZ+LG inhibits aromatase by accelerating mRNA degradation, we report in the present study that TGZ+LG inhibited transcriptional activity of the ArPII in a PPAR-dependent manner. These data reinforce the potential use of synthetic PPAR and RXR agonists for therapeutic applications in diseases in which estrogens, locally or systematically, play prominent pathogenic roles, especially in diseases like breast cancer. In addition, we found that the inhibition disappeared on blockage of NF-B, which was found in turn to positively regulate aromatase. Notably, activation of NF-B has been found involved in the proliferation and metastasis of breast cancer cells (43), for which, although several mechanisms have been suggested, we suppose that stimulation of aromatase might be an additional one. Classically, regulation of ArPII involves PKA-cAMP response element-binding protein (49) and the orphan nuclear receptor steroidogenic factor 1 (Ad4BP/SF-1) (50), but the actual regulation may be much more complicated, at least in that nuclear receptors like PPAR, RXR, and their cross-talk with the transcriptional factor NF-B might also play some important roles.
References
Thompson Jr EA, Siiteri PK 1974 Utilization of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatization of androstenedione. J Biol Chem 249:5364–5372
Means GD, Kilgore MW, Mahendroo MS, Mendelson CR, Simpson ER 1991 Tissue-specific promoters regulate aromatase cytochrome P450 gene expression in human ovary and fetal tissues. Mol Endocrinol 5:2005–2013
Jenkins C, Michael D, Mahendroo M, Simpson E 1993 Exon-specific northern analysis and rapid amplification of cDNA ends (RACE) reveal that the proximal promoter II (PII) is responsible for aromatase cytochrome P450 (CYP19) expression in human ovary. Mol Cell Endocrinol 97:R1–R6
Tsai-Morris CH, Aquilana DR, Dufau ML 1985 Cellular localization of rat testicular aromatase activity during development. Endocrinology 116:38–46
Nitta H, Bunick D, Hess RA, Janulis L, Newton SC, Millette CF, Osawa Y, Shizuta Y, Toda K, Bahr JM 1993 Germ cells of the mouse testis express aromatase. Endocrinology 132:1396–1401
Naftolin F, Ryan KJ, Davies IJ, Reddy VV, Flores F, Petro Z, Kuhn M, White RJ, Takaoka Y, Wolin L 1975 The formation of estrogens by central neuroendocrine tissues. Recent Prog Horm Res 31:295–319
Roselli CE, Horton LE, Resko JA 1985 Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system. Endocrinology 117:2471–2477
Simpson ER, Michael MD, Agarwal VR, Hinshelwood MM, Bulun SE, Zhao Y 1997 Cytochromes P450 11: expression of the CYP19 (aromatase) gene: an unusual case of alternative promoter usage. FASEB J 11:29–36
Bokhman JV 1983 Two pathogenetic types of endometrial carcinoma. Gynecol Oncol 15:10–17
Sasano H, Harada N 1998 Intratumoral aromatase in human breast, endometrial, and ovarian malignancies. Endocr Rev 19:593–607
Yue W, Wang JP, Hamilton CJ, Demers LM, Santen RJ 1998 In situ aromatization enhances breast tumor estradiol levels and cellular proliferation. Cancer Res 58:927–932
Agarwal VR, Bulun SE, Leitch M, Rohrich R, Simpson ER 1996 Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients. J Clin Endocrinol Metab 81:3843–3849
Zhou C, Zhou D, Esteban J, Murai J, Siiteri PK, Wilczynski S, Chen S 1996 Aromatase gene expression and its exon I usage in human breast tumors. J Steroid Biochem Mol Biol 59:163–171
Harada N 1997 Aberrant expression of aromatase in breast cancer tissues. J Steroid Biochem Mol Biol 61:175–184
Bulun SE, Simpson ER 1994 Competitive reverse transcription-polymerase chain reaction analysis indicates that levels of aromatase cytochrome P450 transcripts in adipose tissue of buttocks, thighs, and abdomen of women increase with advancing age. J Clin Endocrinol Metab 78:428–432
Bulun SE, Price TM, Aitken J, Mahendroo MS, Simpson ER 1993 A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription. J Clin Endocrinol Metab 77:1622–1628
Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ, Umesono K, Evans RM 1994 Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc Natl Acad Sci USA 91:7355–7359
Lemberger T, Desvergne B, Wahli W 1996 PPARs: a nuclear receptor-signaling pathway in lipid metabolism. Annu Rev Cell Dev Biol 12:335–363
Tontonoz P, Hu E, Graves RA, Budavari AI, Spiegelman BM 1994 mPPAR 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev 8:1224–1234
Braissant O, Foufelle F, Scotto C, Dauca M, Wahli W 1996 Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-, -?, and - in the adult rat. Endocrinology 137:354–366
Mu YM, Yanase T, Nishi Y, Waseda N, Oda T, Tanaka A, Takayanagi R, Nawata H 2000 Insulin sensitizer, troglitazone, directly inhibits aromatase activity in human ovarian granulosa cells. Biochem Biophys Res Commun 271:710–713
Komar CM, Braissant O, Wahli W, Curry Jr TE 2001 Expression and localization of PPARs in the rat ovary during follicular development and the periovulatory period. Endocrinology 142:4831–4838
Mu YM, Yanase T, Nishi Y, Takayanagi R, Goto K, Nawata H 2001 Combined treatment with specific ligands for PPAR:RXR nuclear receptor system markedly inhibits the expression of cytochrome aromatase in human granulosa cancer cells. Mol Cell Endocrinol 181:239–248
Yanase T, Mu YM, Nishi Y, Goto K, Nomura M, Okabe T, Takayanagi R, Nawata H 2001 Regulation of aromatase by nuclear receptors. J Steroid Biochem Mol Biol 79:187–192
Nishi Y, Yanase T, Mu Y, Oba K, Ichino I, Saito M, Nomura M, Mukasa C, Okabe T, Goto K, Takayanagi R, Kashimura Y, Haji M, Nawata H 2001 Establishment and characterization of a steroidogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor. Endocrinology 142:437–445
Tontonoz P, Hu E, Spiegelman BM 1994 Stimulation of adipogenesis in fibroblasts by PPAR 2, a lipid-activated transcription factor. Cell 79:1147–1156
Rubin GL, Duong JH, Clyne CD, Speed CJ, Murata Y, Gong C, Simpson ER 2003 Ligands for the peroxisomal proliferator-activated receptor and the retinoid X receptor inhibit aromatase cytochrome P450 (CYP19) expression mediated by promoter II in human breast adipose. Endocrinology 143:2863–2871
Wang N, Verna L, Chen NG, Chen J, Li H, Forman BM, Stemerman MB 2002 Constitutive activation of peroxisome proliferator-activated receptor suppresses pro-inflammatory adhesion molecules in human vascular endothelial cells. J Biol Chem 277:34176–34181
Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK 1998 The peroxisome proliferator-activated receptor- is a negative regulator of macrophage activation. Nature 391:79–82
Jiang C, Ting AT, Seed B 1998 PPAR- agonists inhibit production of monocyte inflammatory cytokines. Nature 391:82–86
Natarajan K, Singh S, Burke Jr TR, Grunberger D, Aggarwal BB 1996 Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-B. Proc Natl Acad Sci USA 93:9090–9095
Fujii A, Harada T, Yamauchi N, Iwabe T, Nishi Y, Yanase T, Nawata H, Terakawa N 2003 Interleukin-8 gene and protein expression are up-regulated by interleukin-1? in normal human ovarian cells and a granulosa tumor cell line. Fertil Steril 79:151–157
Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB 1999 NF-B activation by tumor necrosis factor requires the Akt serine-threonine kinase. Nature 401:82–85
Diaz-Meco MT, Berra E, Municio MM, Sanz L, Lozano J, Dominguez I, Diaz-Golpe V, Lain de Lera MT, Alcami J, Paya CV 1993 A dominant negative protein kinase C subspecies blocks NF-B activation. Mol Cell Biol 13:4770–4775
Dunaif A, Scott D, Finegood D, Quintana B, Whitcomb R 1996 The insulin-sensitizing agent troglitazone improves metabolic and reproductive abnormalities in the polycystic ovary syndrome. J Clin Endocrinol Metab 81:3299–3306
Winnett G, van Hagen D, Schrey M 2003 Prostaglandin J2 metabolites inhibit aromatase activity by redox-sensitive mechanisms: potential implications for breast cancer therapy. Int J Cancer 103:600–605
Lovekamp-Swan T, Jetten AM, Davis BJ 2003 Dual activation of PPAR and PPAR by mono-(2-ethylhexyl) phthalate in rat ovarian granulosa cells. Mol Cell Endocrinol 201:133–141
Van Landeghem AA, Poortman J, Nabuurs M, Thijssen JH 1985 Endogenous concentration and subcellular distribution of estrogens in normal and malignant human breast tissue. Cancer Res 45:2900–2906
Grodin JM, Siiteri PK, MacDonald PC 1973 Source of estrogen production in postmenopausal women. J Clin Endocrinol Metab 36:207–214
Bulun SE, Mahendroo MS, Simpson ER 1994 Aromatase gene expression in adipose tissue: relationship to breast cancer. J Steroid Biochem Mol Biol 49:319–326
Zhao Y, Agarwal VR, Mendelson CR, Simpson ER 1996 Estrogen biosynthesis proximal to a breast tumor is stimulated by PGE2 via cyclic AMP, leading to activation of promoter II of the CYP19 (aromatase) gene. Endocrinology 137:5739–5742
Evans CT, Corbin CJ, Saunders CT, Merill JC, Simpson ER, Mendelson CR 1987 Regulation of estrogen biosynthesis in human adipose stromal cells. Effects of dibutyryl cyclic AMP, epidermal growth factor, and phorbol esters on the synthesis of aromatase cytochrome P-450. J Biol Chem 262:6914–6920
Watabe M, Hishikawa K, Takayanagi A, Shimizu N, Nakaki T 2004 Caffeic acid phenethyl ester induces apoptosis by inhibition of NFB and activation of Fas in human breast cancer MCF-7 cells. J Biol Chem 279:6017–6026
Todorov VT, Volkl S, Muller M, Bohla A, Klar J, Kunz-Schughart LA, Hehlgans T, Kurtz A 2004 Tumor necrosis factor- activates NFB to inhibit renin transcription by targeting cAMP-responsive element. J Biol Chem 279:1458–1467
Delerive P, Gervois P, Fruchart JC, Staels B 2000 Induction of IB expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor- activators. J Biol Chem 275:36703–36707
Chen F, Wang M, O’Connor JP, He M, Tripathi T, Harrison LE 2003 Phosphorylation of PPAR via active ERK1/2 leads to its physical association with p65 and inhibition of NF-?. J Cell Biochem 90:732–744
Chung SW, Kang BY, Kim SH, Pak YK, Cho D, Trinchieri G, Kim TS 2000 Oxidized low density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptor- and nuclear factor-B. J Biol Chem 275:32681–32687
Uchimura K, Nakamuta M, Enjoji M, Irie T, Sugimoto R, Muta T, Iwamoto H, Nawata H 2001 Activation of retinoic X receptor and peroxisome proliferator-activated receptor- inhibits nitric oxide and tumor necrosis factor- production in rat Kupffer cells. Hepatology 33:91–99
Michael MD, Michael LF, Simpson ER 1997 A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 (CYP19) gene. Mol Cell Endocrinol 134:147–156
Michael MD, Kilgore MW, Morohashi K, Simpson ER 1995 Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary. J Biol Chem 270:13561–13566(WuQiang Fan, Toshihiko Ya)