Aromatase is the enzyme responsible for the last step of estrogen synthesis. The female hormone, estrogen, is known to stimulate breast cancer cell growth. Because the expression of aromatase in breast cancer tissues is driven by unique promoters I.3 and II, a more complete understanding of the regulatory mechanism of aromatase expression through promoters I.3/II in breast tumors should be valuable in developing targeted therapies, which selectively suppress estrogen production in breast tumor tissue. Results from in vivo footprinting analyses revealed several protein binding sites, numbered 1 to 5. When site 2 (−124/−112 bp, exon I.3 start site as +1) was mutated, promoters I.3/II activity was dramatically reduced, suggesting that site 2 is a positive regulatory element. Yeast one-hybrid screening revealed that a potential protein binding to site 2 was CCAAT/enhancer binding protein δ (C/EBPδ). C/EBPδ was shown to bind to site 2 of aromatase promoters I.3/II in vitro and in vivo. C/EBPδ up-regulated promoters I.3/II activity through this site and, as a result, it also up-regulated aromatase transcription and enzymatic activity. p65, a subunit of nuclear factor-κB (NF-κB) transcription factor, inhibited C/EBPδ–up-regulated aromatase promoters I.3/II and enzymatic activity. This inhibitory effect of p65 was mediated, in part, through prevention of the C/EBPδ binding to site 2. This C/EBPδ binding site in aromatase promoters I.3/II seems to act as a positive regulatory element in non–p65-overexpressing breast cancer epithelial cells, whereas it is possibly inactive in p65 overexpressing cancer epithelial cells, such as estrogen receptor–negative breast cancer cells. [Cancer Res 2008;68(11):4455–64]

Aromatase is the enzyme which catalyzes the last step of estrogen synthesis. It is expressed at higher levels in breast cancer tissue than normal breast tissues (13). The estrogen produced in situ, due to overexpressed aromatase in breast cancer cells, is thought to play a more crucial role in stimulating cancer cell growth than circulating estrogen (4).

The human aromatase gene contains nine translated exons (II–X) and at least 10 tissue-specific untranslated exon Is (I.1, I.2, 2a, I.3, I.4, I.5, I.6, I.7, I.f, and PII). The various exon Is are present at different levels in different aromatase-expressing tissues and cells (57). For example, mRNA found in the placenta mainly contains exon I.1 and mRNA found in the ovary mainly contains exon PII. Each exon I is driven by its own promoter that is located immediately upstream of the corresponding exon I, and each promoter is regulated by different mechanisms. Studies conducted in this and other laboratories have revealed that exons I.3 and PII are the major exon Is in aromatase mRNA isolated from breast cancer tissue, indicating that promoters I.3 and II are the major promoters driving aromatase expression in breast cancer (1, 6, 8, 9). In normal breast stromal cells, promoter I.4 is the major promoter driving aromatase expression (6, 9).

Because the expression of aromatase in breast cancer tissues is driven by unique promoters I.3 and II, a more complete understanding of the regulatory mechanism of aromatase expression through promoters I.3/II in breast tumors should be valuable in developing targeted therapies, which selectively suppress estrogen production in breast cancer tissue. Thus far, promoters I.3/II are regulated by many factors, including follicle-stimulating hormone, cyclic AMP (cAMP), SF-1, cAMP-responsive element binding protein-1 (CREB-1), LRH-1, and BRCA1 (1014). Our laboratory focuses on the regulation of aromatase expression in breast cancer epithelial cells. We first reported that aromatase is expressed in cancerous epithelial cells, as well as stromal cells (15). Although a few subsequent reports showed aromatase is mostly expressed in stromal cells, recent research confirmed that cancerous epithelial cells are an important intratumoral location of aromatase (1619).

Previous research from our laboratory identified two regulatory regions, located near promoters I.3/II (which are ∼200 bp apart from each other) in breast cancer epithelial cells. The first region, S1, is located between the two promoters (20). S1 functions as an enhancer when ERRα-1 binds, but acts as a repressor when COUP-TF, EAR-2, or RAR-γ bind (21, 22). S1 may also function as a positive regulatory element in cancer tissue since ERRα-1 is expressed in most cancer tissue, whereas EAR-2 and RAR-γ are only expressed in a relatively small number of breast cancer tissues. Our findings from molecular studies were recently confirmed by Miki et al. (18), who found a positive correlation between aromatase and ERRα-1 mRNA levels in isolated breast carcinoma cells. The second region found was a cAMP-responsive element (CREaro) located upstream of promoter I.3 TATA box (12). CREaro functions as an enhancer when CREB-1 binds, but acts as a repressor when Snail and Slug bind. However, Snail and Slug are expressed mostly in normal breast tissues, preventing activators from binding to CREaro. This congruently results in a suppression of promoter activity in normal breast tissues (23).

Through in vivo footprinting and yeast one-hybrid screening approaches, our laboratory has now found that CCAAT/enhancer binding protein δ (C/EBPδ) modulates aromatase expression in breast cancer epithelial cells. C/EBPδ is a family member of CCAAT/enhancer binding proteins categorized in basic leucine zipper (bZIP) transcription factors. There are six members of this family: C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ, C/EBPε, and C/EBPζ. Their main role involves regulation of cellular proliferation and differentiation, particularly in hepatocytes, adipocytes, and hematopoietic cells. Among the C/EBP family, C/EBPβ and C/EBPδ are known to have important roles in mammary gland cell function (24). The known roles of C/EBPβ in the mammary gland are cell growth and differentiation, whereas those of C/EBPδ are growth arrest and apoptosis. In preadipocytes, C/EBPβ is reported to bind to the C/EBP binding site in promoter II of aromatase and up-regulate the promoter activity (25). C/EBPδ is expressed in both mammary epithelial cells and stromal cells (26). Furthermore, in this study, p65 was found to inhibit promoters I.3/II and aromatase enzymatic activity up-regulated by C/EBPδ. p65 is also called RelA and is a subunit of nuclear factor-κB (NF-κB) transcription factor. NF-κB family consists of five members: p50 (NF-κB1), p52 (NF-κB2), p65 (RelA), c-rel, and RelB. p65/p50 heterodimers are the most abundant form. In this paper, results are presented to indicate that the C/EBPδ-p65 interaction is another way to regulate aromatase expression in breast cancer cells.

Cell culture. MCF-7, MDA-MB-231, and H295R cells were purchased from American Type Culture Collection. MCF-7 cells were maintained in Eagle's MEM containing 2 mmol/L l-glutamine, 1× penicillin-streptomycin, 1× nonessential amino acid, 1 mmol/L sodium pyruvate, and 10% fetal bovine serum (FBS). MDA-MB-231 cells were maintained in RPMI 1640 supplemented with 2 mmol/L l-glutamine, 1× penicillin-streptomycin, and 10% FBS. H295R cells were maintained in DMEM/F-12 medium supplemented with 2.5 mmol/L l-glutamine, 1 mmol/L sodium pyruvate, 1× penicillin-streptomycin, 2.5% Nu-Serum, and ITS + Premix (BD Biosciences). All the cell lines were cultured at 37°C with 5% CO2.

In vivo footprinting analysis. This analysis allows us to find protein binding sites within the promoter region by comparing in vivo with in vitro samples.

For the in vivo sample, cells were washed twice with PBS and treated with 0.2% dimethyl sulfate (DMS) for 5 min before DNA extraction. Cells were lysed using lysis buffer [0.3 mol/L NaCl, 50 mmol/L Tris-HCl (pH 8), 25 mmol/L EDTA, 0.2% SDS, and 0.2 mg/mL proteinase K] and then incubated for 5 h at 37°C. DNA was extracted by using phenol/chloroform. For the in vitro sample, DNA was extracted from cells prior to DMS treatment. After treatment with 0.125% DMS for 2 min, the reaction was terminated with DMS stop buffer [1.5 mol/L sodium acetate (pH 7), 1 mol/L 2-mercaptoethanol]. Maxam-Gilbert samples were prepared for sequencing (27). All DNA samples were precipitated again and treated with 1 mol/L piperidine. Samples were incubated for 30 min at 90°C and then 10 min on dry ice. After precipitation, the DNA was washed twice with 75% ethanol, speed-vacuum dried overnight, and dissolved in dH2O. Primers were designed for the aromatase promoters I.3/II region: primer 1 (Tm = 59.8), 5′-AGA CAA CTG ATG GAA GGC TCT GAG AAG AC-3′; primer 2 (Tm = 63.4), 5′-CAA CTG ATG GAA GGC TCT GAG AAG ACC TCA ACG-3′; primer 3 (Tm = 65.7), 5′(IR fluorochrome dye 800 labeled)-ATG GAA GGC TCT GAG AAG ACC TCA ACG ATG CCC-3′. Ligation-mediated PCR and direct labeling were performed using a Biomek 2000 liquid-handling robotic workstation and a thermocycler (Beckman Instrument; ref. 28). Electrophoresis (6% sequencing gel) of the sample and scanning were performed in a Li-Cor DNA sequencer (LI-COR). The collected image was visualized by Adobe Photoshop (Adobe Systems).

Plasmid preparation. Aromatase gene promoters I.3/II constructs were prepared by PCR of MCF-7 genomic DNA. The genomic DNA fragments −329/+284 bp and −251/+74 bp of human aromatase promoters I.3/II (exon I.3 start site as +1) were each subcloned into KpnI/XhoI sites of the pGL3-Basic reporter plasmid (Promega). C/EBPδ and p65 expression plasmids were prepared by inserting the coding sequence of C/EBPδ and p65, respectively, cloned from the yeast one-hybrid screening into a pCIneo plasmid (Promega). Accuracy of plasmids was confirmed by sequencing.

Yeast one-hybrid screening. Yeast one-hybrid screening was performed according to the manufacturer's protocol MATCHMAKER One-Hybrid System (Clontech Laboratories). Three repeats of 20-bp DNA sequence of site 2 were used as a bait. A human mammary gland library was purchased from Clontech.

Transient transfection and reporter gene assay. Cells were transfected with pGL3-Basic-pI.3/II reporter plasmids (−251/+74 or −329/+284 bp) using Lipofectamine 2000 (Invitrogen), as described previously (29). The total amount of DNA (per well) used for cotransfection was 2 μg. The relative luciferase activity was calculated by dividing the light unit of luciferase activity by the protein concentration of each sample.

Gel mobility shift assay. C/EBPδ protein was obtained from in vitro transcription/translation by using TNT Quick Coupled Transcription/Translation Systems (Promega) according to the manufacturer's protocol. The DNA sequence from aromatase promoters I.3/II −165/−80 bp (containing sites 1, 2, and 3) were end-labeled with [γ-32P]ATP. One microgram of DNA fragment was incubated at 37°C for 45 min with 1× kinase buffer, T4 kinase, and 0.02 mCi [32P]ATP (MP Biomedicals). One microliter of 0.5 mol/L EDTA was added to terminate the reaction. This labeled fragment was used as a probe after purification with Sephadex G25 Column (Roche Applied Science) and diluted into 9,000 cpm/μL. The protein was then incubated on ice for 1-h in a mixture containing 1× binding buffer, 0.2 μg of poly(dI-dC) (Amersham Biosciences), and competitors. 10× binding buffer consists of 0.1 mol/L Tris-HCl (pH 7.5), 0.5 mol/L NaCl, 10 mmol/L MgCl2, 5 mmol/L EDTA, 5 mmol/L DTT, and 40% glycerol. Three different unlabeled fragments were used as competitors: aromatase promoters I.3/II −165/−80 bp fragment, 30-bp of the site 2 sequence, and a completely different sequence (30-bp of the site 5 sequence). The mixture was then incubated on ice for another hour with 1 μL of the diluted 32P-labeled probe. Two microliters of 10× gel loading buffer [250 mmol/L Tris-HCl (pH 7.5), 0.2% bromophenol blue, and 40% glycerol] were added to each reaction mixture before being electrophoresed onto a 4% acrylamide/bis-acrylamide (59:1) gel. After drying, the gel was exposed to Basic Autorad Film (ISC Bioexpress) and developed using a Konica SRX-101A (Konica). To eliminate the possibility of nonspecific binding of protein in rabbit reticulocyte lysate to the hot probe, lysate control (included in TNT Quick Coupled Transcription/Translation kit) was also run on the gel.

Chromatin immunoprecipitation assay. Chromatin immunoprecipitation (ChIP) assays were performed according to the manufacturer's protocol using a kit purchased from Upstate. DNA samples were sonicated using Digital Sonifier (Branson). Immunoprecipitation was performed with normal rabbit IgG (Santa Cruz Biotechnology) or anti-C/EBPδ antibody (Santa Cruz Biotechnology). DNA (1 μL), dissolved in 50 μL of TE, was subjected to PCR amplification in a 25-μL reaction mixture containing 0.25 units of ChromaTaq DNA polymerase (Denville Scientific, Inc.), 1× ChromaTaq reaction buffer, 0.2 μmol/L of forward/reverse primers, 2 mmol/L of MgCl2, and 0.2 mmol/L of deoxynucleotide triphosphate mix. Primers were designed to flank the C/EBPδ binding site in promoters I.3/II of aromatase (forward 5′-TCAACGATGCCCAAGAAATG-3′, reverse 5′-CATTCCCAATTGAAAGCCAAA-3′). For ChIP assay in Fig. 5D, H295R cells were transfected with p65 expression plasmid (empty plasmid as a control) prior to the assay. ChIP assay was performed after a 24-h posttransfection incubation.

Western blotting. MCF-7 and H295R cells were transfected with C/EBPδ and/or p65 expression plasmids (empty plasmid as a control). After a 24-h incubation after the transfection, cells were lysed and applied for Western blotting using anti-C/EBPδ antibody (Santa Cruz Biotechnology) or p65 antibody (Cell Signaling Technology), as described previously (29). The blot was reprobed with anti-actin (Santa Cruz Biotechnology) as a loading control.

RNA isolation and semiquantitative Exon I–specific reverse transcription–PCR analysis. MCF-7 and H295R cells were transfected with C/EBPδ expression plasmid or empty plasmid as a control. After a 24-h incubation after the transfection, total RNA was isolated by using TRIZOL reagent (Invitrogen) according to the manufacturer's protocol. The procedures of reverse transcription and aromatase exon I–specific PCR were previously described (29). As an internal control to normalize aromatase mRNA expression in each sample, a set of human β-actin–specific primers was used. Each PCR product was electrophoresed on a 1.8% agarose gel and stained with ethidium bromide.

“In-cell” aromatase assay. Transient transfection of C/EBPδ, p65 expression plasmids, and/or empty plasmid into H295R cells was performed, as described above. Aromatase activity was measured in a 3H2O release assay, as previously described (30), after 24 h of incubation after transfection.

New protein binding sites in promoters I.3/II of aromatase by in vivo footprinting. To find new regulatory sites in aromatase promoters I.3/II, as well as to verify the previously shown regulatory elements (S1 and CREaro), in vivo footprinting analyses were performed. The experiments were conducted with both estrogen receptor (ER)–positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. The result showed several footprinted DNA regions (Fig. 1). MCF-7 and MDA-MB-231 cells had a similar pattern. Clear footprints were seen on the previously reported regulatory element, CREaro, and promoter I.3 TATA box, demonstrating that this technique is useful for the identification of transcriptional regulatory sites. There was no clear footprint in S1 region, probably because the position of the primers was too far from S1 for a clear footprint. By reviewing our results, we decided to focus on five sites with the clearest footprints. They were named site 1 to site 5. The actual positions of the five sites are shown in Fig. 2A.

Figure 1.

In vivo footprinting analysis of aromatase promoters I.3/II. Marker (M), in vivo sample (v), in vitro sample (vt), G, G+A, T+C, C: Maxam-Gilbert samples for the sequencing. The samples were run on a 6% polyacrylamide sequencing gel, and the collected image was visualized using Adobe Photoshop. Several footprinted sites in the aromatase promoters I.3/II region were identified. If footprints were identified in the in vivo samples, but not the in vitro samples, the footprinted regions were thought to associate with protein binding that protects DNA from DMS treatment in vivo. Five regions (sites 1–5) were selected for functional analysis: sites 1 to 3 are located upstream of promoter I.3 TATA box, whereas sites 4 and 5 are located downstream of promoter I.3 TATA box. The numbers on the right side of the two gel images correspond to the numbering shown in Fig. 2A (exon I.3 start site as +1).

Figure 1.

In vivo footprinting analysis of aromatase promoters I.3/II. Marker (M), in vivo sample (v), in vitro sample (vt), G, G+A, T+C, C: Maxam-Gilbert samples for the sequencing. The samples were run on a 6% polyacrylamide sequencing gel, and the collected image was visualized using Adobe Photoshop. Several footprinted sites in the aromatase promoters I.3/II region were identified. If footprints were identified in the in vivo samples, but not the in vitro samples, the footprinted regions were thought to associate with protein binding that protects DNA from DMS treatment in vivo. Five regions (sites 1–5) were selected for functional analysis: sites 1 to 3 are located upstream of promoter I.3 TATA box, whereas sites 4 and 5 are located downstream of promoter I.3 TATA box. The numbers on the right side of the two gel images correspond to the numbering shown in Fig. 2A (exon I.3 start site as +1).

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Figure 2.

Functional characterization of sites 1 to 5 by DNA mutagenesis experiments. A, the fragment between the arrows (−251/+74 or −329/+284 bp; exon I.3 start site as +1) was inserted into pGL3-Basic. Five separate mutants (M1–M5), which had been mutated on only one of the five sites, were generated by PCR using primers with the desired mutations. The mutant label indicates which site was mutated. The changes in the sequence of each site (bold uppercase) is indicated by the bases directly below them. TATA boxes of promoter I.3 and promoter II, exon I.3, exon PII, exon II, aromatase coding sequence (CDS), CREaro, and S1 are indicated in the figure. B, the pGL3-Basic wild-type (Wt) and M1-M5 plasmids were transiently transfected into MCF-7 and H295R cells using Lipofectamine 2000. After transfection, the cells were incubated for 24 h and then lysed. Aliquots of the lysate were used for luciferase assay. The relative luciferase activity was obtained by normalization with protein concentrations. Statistically significant differences compared with wild-type were indicated as * for P < 0.0002. Statistical analyses were performed using the two-tailed Student's t test.

Figure 2.

Functional characterization of sites 1 to 5 by DNA mutagenesis experiments. A, the fragment between the arrows (−251/+74 or −329/+284 bp; exon I.3 start site as +1) was inserted into pGL3-Basic. Five separate mutants (M1–M5), which had been mutated on only one of the five sites, were generated by PCR using primers with the desired mutations. The mutant label indicates which site was mutated. The changes in the sequence of each site (bold uppercase) is indicated by the bases directly below them. TATA boxes of promoter I.3 and promoter II, exon I.3, exon PII, exon II, aromatase coding sequence (CDS), CREaro, and S1 are indicated in the figure. B, the pGL3-Basic wild-type (Wt) and M1-M5 plasmids were transiently transfected into MCF-7 and H295R cells using Lipofectamine 2000. After transfection, the cells were incubated for 24 h and then lysed. Aliquots of the lysate were used for luciferase assay. The relative luciferase activity was obtained by normalization with protein concentrations. Statistically significant differences compared with wild-type were indicated as * for P < 0.0002. Statistical analyses were performed using the two-tailed Student's t test.

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Functional characterization of the newly identified five sites. To investigate whether the five newly identified sites are functionally significant for aromatase promoters I.3/II activity, DNA mutagenesis experiments were performed. The human aromatase promoters I.3/II region (−251/+74 bp) was inserted into the multiple cloning site of the pGL3-Basic plasmid (wild-type). After the wild-type promoter activity was shown (four to five times higher than pGL3-Basic empty reporter activity), five separate mutants were prepared (M1–M5), each of which was mutated on only one of its five sites (Fig. 2A). The mutant label indicates which site was mutated. For example, the site 1 mutant (M1) contained a changed sequence in the first site only. Human adrenocortical carcinoma cell line H295R was also used for this experiment, besides MCF-7, because it contains high levels of aromatase whose expression is driven by promoters I.3/II and gives us higher luciferase activity after transfection. Our analysis revealed that only M2 had a significantly lower luciferase activity (similar to the pGL3-Basic empty reporter activity) when compared with the wild-type in both MCF-7 and H295R cells (Fig. 2B), indicating that the protein binding to site 2 was an activator and site 2 is a positive regulatory element. Mutations in other sites did not change luciferase activity compared with the control.

Site 2 binding protein C/EBPδ. Yeast one-hybrid screening was performed to obtain candidate DNA binding protein for site 2. The screening was performed using an activation domain (AD) fusion human mammary gland library. Seventy-eight colonies were obtained from site 2 screening. Among the clones, 30% of them were C/EBPδ. Interestingly, our screening of the AD fusion human mammary gland library did not isolate other CCAAT/enhancer binding proteins. Besides C/EBPδ, immediate early response 2 protein was identified from two clones. However, it did not have any regulatory effects on promoters I.3/II (transfection experiments; data not shown). The remaining candidate proteins were each found in only one clone. The result of sequence analysis using Transcription Element Search System (TESS)1

showed site 2 (−124/−112 bp; ttgTtttGaAAtt) as a C/EBP binding site (data not shown), which supports the yeast one-hybrid screening result.

To confirm C/EBPδ binding to site 2, gel mobility shift assay and ChIP assay were conducted. The DNA sequence from aromatase promoters I.3/II −165/−80 bp (contains sites 1, 2, and 3) was used as a hot probe (labeled with 32P) for the gel mobility shift assay. C/EBPδ protein was generated from in vitro transcription and translation of C/EBPδ expression plasmid. In vitro translated C/EBPδ protein was verified on a 12% polyacrylamide gel (data not shown). The results showed that C/EBPδ clearly binds to the aromatase promoters I.3/II −165/−80 bp region (Fig. 3A). The unlabeled −165/−80 bp fragment and 30-bp site 2 fragment competed with the labeled probe, whereas the 30-bp site 5 fragment (used as a control competitor of totally different sequence from site 2) did not, suggesting the interaction of C/EBPδ with DNA around the site 2 region of aromatase promoters I.3/II is sequence-specific in vitro. The binding of C/EBPδ to site 2 was also confirmed in vivo by ChIP assay (Fig. 3B). Cell lysates were obtained from MCF-7 and H295R cells and immunoprecipitated with anti-C/EBPδ antibody (with normal rabbit IgG as a control). Band intensity for DNA precipitated by C/EBPδ antibody was significantly stronger than that for DNA precipitated by the control IgG in both MCF-7 and H295R cells, indicating binding of C/EBPδ to the site 2 region in vivo.

Figure 3.

C/EBPδ binding to site 2. A, the 35S-labeled C/EBPδ was treated with 1 mmol/L CaCl2. The −165/−80 bp fragment was 32P-labeled and incubated with the C/EBPδ protein. Unlabeled competitors (−165/−80 bp fragment, site 2, or site 5) were also added to the mixture. Samples were electrophoresed onto a 4% acrylamide/bis-acrylamide (59:1) gel. Arrow, complex of C/EBPδ and −165/−80 bp fragment. B, cell lysates obtained from MCF-7 and H295R cells were immunoprecipitated with normal rabbit IgG (control) and anti-C/EBPδ antibody after cross-linking with 1% formaldehyde. PCR was performed using primers to amplify site 2 region of promoters I.3/II.

Figure 3.

C/EBPδ binding to site 2. A, the 35S-labeled C/EBPδ was treated with 1 mmol/L CaCl2. The −165/−80 bp fragment was 32P-labeled and incubated with the C/EBPδ protein. Unlabeled competitors (−165/−80 bp fragment, site 2, or site 5) were also added to the mixture. Samples were electrophoresed onto a 4% acrylamide/bis-acrylamide (59:1) gel. Arrow, complex of C/EBPδ and −165/−80 bp fragment. B, cell lysates obtained from MCF-7 and H295R cells were immunoprecipitated with normal rabbit IgG (control) and anti-C/EBPδ antibody after cross-linking with 1% formaldehyde. PCR was performed using primers to amplify site 2 region of promoters I.3/II.

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Up-regulation of aromatase by C/EBPδ. To determine the functional role of C/EBPδ on aromatase promoters I.3/II, transient transfection experiments were performed using aromatase promoters I.3/II luciferase reporter plasmid and C/EBPδ expression plasmid. MCF-7 and H295R cells were used for this experiment. Overexpressed C/EBPδ protein levels after transfection were confirmed by Western blotting (Fig. 4A). C/EBPδ failed to up-regulate the promoter activity when site 2 was mutated, whereas mutations in other sites did not abolish the ability of C/EBPδ as an activator of promoters I.3/II (Fig. 4B). This suggested that C/EBPδ enhances promoters I.3/II activity through site 2, agreeing with the previous result of mutagenesis experiment in which site 2 was an enhancer element (Fig. 2B).

Figure 4.

Up-regulation of aromatase by C/EBPδ. A, Western blotting of C/EBPδ (actin as a loading control) was performed with MCF-7 and H295R cells that were transfected with C/EBPδ expression plasmid or empty plasmid. B, six different reporter plasmids were used: wild-type (pGL3-Basic −251/+74 bp) and five separate mutants (M1–M5) that were each mutated on only one of its five sites. The plasmids were transfected into MCF-7 and H295R cells using Lipofectamine 2000. After transfection, the cells were incubated for 24 h and then lysed. Aliquots of the lysate were used for luciferase assay. The relative luciferase activity was obtained by normalization with protein concentrations. Statistically significant differences compared with empty plasmid transfectants were indicated as * for P < 0.0002. Statistical analyses were performed using the two-tailed Student's t test. C, H295R cells were transfected with C/EBPδ expression plasmid or empty plasmid. After 24-h incubation after the transfection, total RNA was isolated and used for exon I–specific reverse transcription–PCR. Exons I.1, I.4, I.6, I.3, PII, and II specific primers were used. β-Actin was amplified as an internal control. D, H295R cells were transfected with C/EBPδ expression plasmid or empty plasmid. After 24-h incubation after transfection, in-cell aromatase assay was performed. Significant difference was indicated as * for P < 0.001 compared with parental H295R cells or empty plasmid-transfected H295R cells.

Figure 4.

Up-regulation of aromatase by C/EBPδ. A, Western blotting of C/EBPδ (actin as a loading control) was performed with MCF-7 and H295R cells that were transfected with C/EBPδ expression plasmid or empty plasmid. B, six different reporter plasmids were used: wild-type (pGL3-Basic −251/+74 bp) and five separate mutants (M1–M5) that were each mutated on only one of its five sites. The plasmids were transfected into MCF-7 and H295R cells using Lipofectamine 2000. After transfection, the cells were incubated for 24 h and then lysed. Aliquots of the lysate were used for luciferase assay. The relative luciferase activity was obtained by normalization with protein concentrations. Statistically significant differences compared with empty plasmid transfectants were indicated as * for P < 0.0002. Statistical analyses were performed using the two-tailed Student's t test. C, H295R cells were transfected with C/EBPδ expression plasmid or empty plasmid. After 24-h incubation after the transfection, total RNA was isolated and used for exon I–specific reverse transcription–PCR. Exons I.1, I.4, I.6, I.3, PII, and II specific primers were used. β-Actin was amplified as an internal control. D, H295R cells were transfected with C/EBPδ expression plasmid or empty plasmid. After 24-h incubation after transfection, in-cell aromatase assay was performed. Significant difference was indicated as * for P < 0.001 compared with parental H295R cells or empty plasmid-transfected H295R cells.

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To determine whether C/EBPδ enhances transcription of aromatase and then aromatase enzymatic activity as a consequence of up-regulation of aromatase promoter activity, C/EBPδ expression plasmid or empty plasmid was transfected into H295R cells. H295R cells were chosen for this experiment because this cell line contains measurable aromatase whose expression is driven by promoters I.3/II. The levels of mRNA containing exons I.3, PII, and II were increased by C/EBPδ overexpression, suggesting that C/EBPδ increased aromatase total mRNA level through promoters I.3/II (Fig. 4C). Overexpression of C/EBPδ also increased aromatase activity in H295R cells, compared with parental H295R cells or empty plasmid-transfected H295R cells (P < 0.001; Fig. 4D). These results indicate that C/EBPδ up-regulates promoters I.3/II activity, resulting in up-regulation of aromatase expression and then aromatase activity.

Inhibitory effect of p65 on C/EBPδ–up-regulated aromatase promoters I.3/II activity. Our yeast one-hybrid screening indicated that p65 was a candidate protein that binds to site 4 and site 5 (data not shown). Furthermore, sequence analysis using TESS revealed that site 1 contained a p65 binding sequence (data not shown). Although p65 itself did not show any effect on aromatase (as shown as controls in Fig. 5B and C), the possibility of p65 involvement was high because of the results from yeast one screening and TESS. We investigated whether p65 has any effect on C/EBPδ function on aromatase promoters I.3/II. p65 expression plasmid was cotransfected with empty and/or C/EBPδ expression plasmids into MCF-7 and H295R cells. The reporter plasmid used for the experiments was pGL3-Basic −329/+284 bp. Overexpressed C/EBPδ and p65 protein levels after transfection were confirmed by Western blotting (Fig. 5A). In the absence of p65 overexpression, C/EBPδ up-regulated aromatase promoters I.3/II activity in both MCF-7 cells (P = 0.05) and H295R cells (P < 0.0001), as shown previously (Fig. 5B). However, when p65 was overexpressed, the increased promoters I.3/II activity by C/EBPδ dropped to 24% of the activity in the absence of p65 in MCF-7 cells (P = 0.038) and to 9% in H295R cells (P < 0.0001). Figure 5B also shows that p65 does not contain any effect on promoters I.3/II activity by itself. This indicates that p65 can inhibit aromatase promoters I.3/II activity only when the activity is up-regulated by C/EBPδ.

Figure 5.

Inhibitory effect of p65 on C/EBPδ–up-regulated aromatase through destabilizing binding of C/EBPδ to site 2. A, Western blotting of C/EBPδ and p65 (actin as a loading control) was performed with MCF-7 and H295R cells that were cotransfected with C/EBPδ or/and p65 expression plasmids (empty plasmid as a control). B, p65 expression plasmid was cotransfected with empty and C/EBPδ expression plasmids into MCF-7 and H295R cells. The reporter plasmid used in the experiments was pGL3-Basic −329/+284 bp. Statistical analyses were performed using the two-tailed Student's t test. C, H295R cells were cotransfected with expression plasmids (C/EBPδ and/or p65) and empty plasmid as a control. After 24-h incubation after transfection, aromatase assay was performed. Statistical analyses were performed using two-tailed Student's t test. D, ChIP assay was performed with H295R cells that were transfected with p65 expression plasmid or empty plasmid. Immunoprecipitation was conducted with normal rabbit IgG or anti-C/EBPδ antibody. PCR was performed using primers to amplify site 2 region of promoters I.3/II.

Figure 5.

Inhibitory effect of p65 on C/EBPδ–up-regulated aromatase through destabilizing binding of C/EBPδ to site 2. A, Western blotting of C/EBPδ and p65 (actin as a loading control) was performed with MCF-7 and H295R cells that were cotransfected with C/EBPδ or/and p65 expression plasmids (empty plasmid as a control). B, p65 expression plasmid was cotransfected with empty and C/EBPδ expression plasmids into MCF-7 and H295R cells. The reporter plasmid used in the experiments was pGL3-Basic −329/+284 bp. Statistical analyses were performed using the two-tailed Student's t test. C, H295R cells were cotransfected with expression plasmids (C/EBPδ and/or p65) and empty plasmid as a control. After 24-h incubation after transfection, aromatase assay was performed. Statistical analyses were performed using two-tailed Student's t test. D, ChIP assay was performed with H295R cells that were transfected with p65 expression plasmid or empty plasmid. Immunoprecipitation was conducted with normal rabbit IgG or anti-C/EBPδ antibody. PCR was performed using primers to amplify site 2 region of promoters I.3/II.

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Next, we investigated whether p65 overexpression can repress up-regulated aromatase enzymatic activity by C/EBPδ. H295R cells were cotransfected with C/EBPδ and/or p65 expression plasmids and empty plasmid. After 24-h of incubation after transfection, aromatase assay was performed. In the absence of p65 overexpression, C/EBPδ up-regulated aromatase activity (Fig. 5C). This up-regulation was abolished in the presence of p65 overexpression (P < 0.0002).

Prevention of C/EBPδ binding to site 2 by p65. Although sites 1, 4, and 5 were the candidate binding sites for p65, none of the three sites were shown to be involved in the inhibitory effect of p65 (data not shown). There could, however, be more p65 binding sites in promoters I.3/II, other than sites 1, 4, and 5. It is also possible that p65 binds directly to C/EBPδ on site 2, because p65 and C/EBPδ physically interact with each other. We hypothesized that p65 binds to C/EBPδ on site 2 and modulates C/EBPδ transactivation function. p65 might possibly destabilize C/EBPδ binding to site 2. To investigate this possibility, we performed ChIP assay using empty or p65 plasmid-transfected H295R cells. Immunoprecipitation was conducted with anti-C/EBPδ antibody or normal rabbit IgG (as a negative control). The band intensity for DNA precipitated by C/EBPδ antibody was significantly stronger than that of DNA precipitated by control IgG in empty plasmid-transfected H295R cells (Fig. 5D). This indicates that C/EBPδ binds to site 2 in vivo. However, the band intensity for DNA precipitated by C/EBPδ antibody was similar with that of DNA precipitated by control IgG in p65 expression plasmid-transfected H295R cells, indicating that p65 overexpression disrupted C/EBPδ binding to site 2 in vivo.

This is the first time in vivo protein binding was visualized on human aromatase promoters I.3/II. Several new protein binding regions, named site 1 to site 5, in the promoters were found. The mutagenesis experiment indicated that none of the sites, with the exception of site 2 (positive regulatory element), modulated promoters I.3/II activity, although protein binding to the sites was clearly shown through in vivo footprinting analysis. This raised the question of whether it was possible that the proteins binding to the sites were not transcriptional factors. We do not have a clear answer for this question at this moment.

TESS searches DNA binding proteins whose binding site sequences match your sequences of interest. TESS results showed the sequence of site 2 matches with C/EBP binding site, which supports the result of our yeast one-hybrid screening. Zhou et al. also reported the aromatase promoters I.3/II −350/−337 bp region (exon PII start site as +1 in their numbering), which is our site 2 region, as C/EBP binding sites in preadipocytes (25). This strongly supports that our yeast one-hybrid screening for site 2 was a success.

C/EBPδ was found to bind to site 2 (−124/−112 bp; exon I.3 start site as +1) of aromatase promoters I.3/II and up-regulate the promoter activity. Furthermore, p65 inhibits the positive regulatory function of C/EBPδ, in part through destabilizing C/EBPδ binding to site 2. Breast cancer tissues contain higher levels of aromatase than normal breast tissues. If the up-regulated expression of aromatase in breast cancer tissue is due to C/EBPδ, this factor may be expressed at higher levels in breast cancer tissues than normal breast tissues. Although loss of function in C/EBPδ gene expression has been reported in human breast cancer epithelial cells (due to the function of C/EBPδ in growth arrest and apoptosis; ref. 31), alteration of C/EBPδ expression level in breast cancer tissue is controversial. Decreased level of C/EBPδ mRNA in mammary epithelial cells was reported as the disease develops from normal to DCIS, invasive, and then metastatic breast cancer, although the sample numbers are small (two to eight samples; refs. 32, 33). Tang et al. showed that 32% (18 samples of 57) of primary breast tumor tissue (mixture of epithelial and stromal cells) showed decreased mRNA level of C/EBPδ (34). However, these results can also be interpreted, as showing the majority of the breast tumor samples do not exhibit reduced level of C/EBPδ. On the other hand, there are also reports showing up-regulation of C/EBPδ expression in breast cancer epithelial and stromal cells. Meng et al. reported the treatment of mouse fibroblast 3T3-L1 cells with T47D cell condition medium increased the mRNA levels of C/EBPβ and C/EBPδ (35). Milde-Langosch et al. showed C/EBPδ protein expression in breast cancer epithelial cell lines was up-regulated 6.3% to 15.5% of the control (26). In the carcinomas (mixture of epithelial and stromal cells), C/EBPδ expression was elevated at a varying rate of 0 to 191%. Taken all together, C/EBPδ expression levels in breast cancer tissues vary from sample to sample. Therefore, the up-regulated expression of aromatase in breast cancer tissue can be due to C/EBPδ in cells expressing this protein.

Zhou et al. reported that T47D breast cancer cell–conditioned medium induced C/EBPβ expression in human adipose fibroblast cells and that C/EBPβ enhanced aromatase expression by binding to a promoter II regulatory element (25). There are at least two C/EBP binding sites in promoters I.3/II. The ones reported by Zhou et al. are −350/−337 and −317/−304 bp (based on their numbering method), which are located in site 2 and site 3, respectively, in our study. C/EBPδ (not C/EBPβ) was identified from a yeast one-hybrid screening of an AD fusion human mammary gland library and found as an enhancer of promoters I.3/II in our study. This suggests that C/EBPβ is important for aromatase regulation in preadipocytes, whereas C/EBPδ is important in cancerous epithelial cells. The same group also reported C/EBPβ forms a complex with active activating transcription factor-2 and CBP on −317/−304 bp of promoters I.3/II and up-regulates the promoter activity (36). CBP is a coactivator that contains histone acetyltransferase activity and acetylates histones. It serves as a mediator protein that brings transcriptional activators, such as CREB, and basal transcription machinery together. McCauslin et al. recently revealed that overexpression of PKA, with the combination with C/EBPδ, up-regulated nerve growth factor (NGF) gene promoter activity and that C/EBPδ and CREB bind to the promoter (37). It is possible that C/EBPδ also forms a complex with CBP and CREB (phosphorylated by PKA) and up-regulates promoter activity (NGF promoter and aromatase promoters I.3/II), because C/EBPδ actually interacts with CBP and helps CBP phosphorylation (38, 39).

C/EBPδ has been reported to also interact with ER. In the presence of estrogen, C/EBPδ physically interacts with ER that is binding to estrogen, resulting in the inhibition of C/EBPδ function on insulin-like growth factor I gene activation in osteoblasts (40). On the contrary, another group reported that ER (in the presence of estrogen) can function as a mediator protein that connects C/EBPβ and Sp1 on human prolactin receptor gene promoter, resulting in up-regulation of the promoter activity (41). Hence, we were interested in the effect of estrogen (via ER) on C/EBPδ and aromatase promoters I.3/II activity. However, we did not see any effect of estrogen on C/EBPδ-mediated aromatase expression (data not shown).

Although p65 itself did not show any effect on aromatase (Fig. 5), it inhibited C/EBPδ function on aromatase promoters I.3/II. p65 and C/EBPδ are reported to interact with each other through Rel homology domain of p65 and bZIP domain of C/EBPδ (42, 43). When C/EBPδ binds to C/EBP binding site and p65 binds to NF-κB binding site, they can interact and may bend DNA to form a loop so that the transcription factor complex will be closer to the basal transcription machinery, resulting in up-regulation of the promoter activity (42, 44). C/EBPδ can also bind to p65 on NF-κB binding site and p65 can bind to C/EBPδ on C/EBP binding site, modulating promoter activity (4345). Our study implied that p65 binds to C/EBPδ on C/EBP binding site of aromatase promoters I.3/II and destabilizes the C/EBPδ binding, leading to the down-regulation of enhanced promoter activity by C/EBPδ.

NF-κB is reported to be constitutively active in ER-negative, hormone-independent breast cancer tissue (both in epithelial and stromal cells). Nakshatri et al. compared the NF-κB activity in ER-positive and ER-negative breast cancer epithelial cell lines (46). All the ER-negative cell lines (also ER-negative poorly differentiated primary breast tumors) contained constitutive NF-κB activation. Interestingly, it was also shown that activated NF-κB level was reduced when estrogen was reintroduced to the MCF-7 cells, which had been cultured in estrogen-depleted medium (47). This indicates the involvement of estrogen-ER in the regulation of NF-κB activation. The constitutively active NF-κB in ER-negative breast cancer could be due to the reduced IκB expression or reduced ER expression. Both IκB and ER contain inhibitory effects on NF-κB activity. The active NF-κB blocks tumor necrosis factor-α–induced, ionizing radiation–induced, and chemotherapeutic agent–induced apoptosis (46) and stimulates cell proliferation, resulting in tumor growth (48). The subunit of NF-κB that we investigated in our study is p65/RelA. Both p65 and p50 were reported to be activated in ER-negative breast cancer (47, 49, 50). There was no correlation between C/EBPδ expression level and ER expression status, indicating that C/EBPδ is expressed at similar levels in ER-positive and ER-negative breast cancers (26). Aromatase expression mediated by C/EBPδ is possibly reduced when breast cancer develops from ER-positive to the ER-negative phenotype (Fig. 6). This could be due to NF-κB being constitutively active in ER-negative breast cancer epithelial cells, which results in destabilization of C/EBPδ binding to its site, which in turn down-regulates aromatase promoters I.3/II activity. Lower expression of aromatase in ER-negative breast cancer epithelial cells (compared with ER-positive breast cancer epithelial cells) was reported (18). Although it is possible that the decreased expression level of aromatase in ER-negative breast cancer epithelial cells is partially due to the down-regulation of promoters I.3/II activity through the inhibitory effect of p65 on C/EBPδ, it should be emphasized that regulation of aromatase expression may be more complex than what we have discussed here.

Figure 6.

Model of C/EBPδ and p65 function on aromatase expression in normal and breast cancer epithelial cells. Higher expression of aromatase in breast cancer epithelial cells can be partially mediated by site 2 (−124/−112 bp)–C/EBPδ interaction. In ER-positive breast cancer epithelial cells, estrogen produced by overexpressed aromatase stimulates cancer cell growth through the ER pathway. Aromatase expression mediated by C/EBPδ is possibly reduced when breast cancer develops from ER-positive to the ER-negative phenotype due to constitutively active p65 in ER-negative breast cancer epithelial cells. The constitutively active p65 in ER-negative breast cancer epithelial cells could destabilize C/EBPδ binding to its binding site, which in turn down-regulates aromatase promoters I.3/II activity.

Figure 6.

Model of C/EBPδ and p65 function on aromatase expression in normal and breast cancer epithelial cells. Higher expression of aromatase in breast cancer epithelial cells can be partially mediated by site 2 (−124/−112 bp)–C/EBPδ interaction. In ER-positive breast cancer epithelial cells, estrogen produced by overexpressed aromatase stimulates cancer cell growth through the ER pathway. Aromatase expression mediated by C/EBPδ is possibly reduced when breast cancer develops from ER-positive to the ER-negative phenotype due to constitutively active p65 in ER-negative breast cancer epithelial cells. The constitutively active p65 in ER-negative breast cancer epithelial cells could destabilize C/EBPδ binding to its binding site, which in turn down-regulates aromatase promoters I.3/II activity.

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In summary, this newly identified C/EBPδ binding site (−124/−112 bp) is the third regulatory element (S1 and CREaro are the first and the second) of human aromatase promoters I.3/II in breast cancer epithelial cells found in our laboratory. C/EBPδ up-regulates breast cancer–specific aromatase promoters I.3/II through this site by directly binding to it and p65 inhibits the positive regulatory function of C/EBPδ in breast cancer epithelial cells. This C/EBPδ binding site in aromatase promoters I.3/II seems to act as a positive regulatory element in non–p65-overexpressing breast cancer epithelial cells, whereas it is possibly inactive in p65 overexpressing cancer epithelial cells, such as ER-negative breast cancer cells.

Grant support: California Breast Cancer Research Program Dissertation award CBCRP-10GB-0088 (I. Kijima) and NIH grants CA44735 and ES08258 (S. Chen).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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