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(American Journal of Pathology. 2004;165:2019-2031.)
© 2004 American Society for Investigative Pathology

E4F1, a Novel Estrogen-Responsive Gene in Possible Atheroprotection, Revealed by Microarray Analysis

Yasuhiro Nakamura*{dagger}, Katsuhide Igarashi{ddagger}, Takashi Suzuki*, Jun Kanno{ddagger}, Tohru Inoue§, Chika Tazawa*, Masayuki Saruta*, Tomoko Ando{ddagger}, Noriko Moriyama{ddagger}, Toru Furukawa, Masao Ono*, Takuya Moriya*, Kiyoshi Ito||, Haruo Saito{dagger}, Tadashi Ishibashi{dagger}, Shoki Takahashi{dagger}, Shogo Yamada{dagger} and Hironobu Sasano*

From the Departments of Pathology,* Radiology,{dagger} Molecular Pathology, and Gynecology,|| Tohoku University School of Medicine, Sendai; and the Division of Toxicology{ddagger} and the Biological Safety Research Center,§ National Institute of Health Sciences, Tokyo, Japan


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Estrogen has been postulated to be involved in inhibition of vascular smooth muscle cell (VSMC) proliferation mainly via estrogen receptor (ER), but the detailed mechanism has remained primarily unknown. Therefore, in this study, microarray analysis was used in two types of cultured human VSMCs: one positive for ER{alpha}, and the other for ERß, which were treated by estrogens to detect the estrogen-responsive genes. We also used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate mRNA levels of selective target gene (TG) in these cells. We further studied whether the TG product was involved in inhibition of proliferation using small interfering RNA (siRNA) of the TG transfection. We subsequently used quantitative RT-PCR and in situ hybridization analysis to evaluate the expression of these gene products in human aorta. E4F1, a possible inducer of cell growth arrest, was markedly increased only in ER{alpha}-positive VSMCs by estrogens in both microarray and RT-PCR analyses. Blocking of E4F1 using siRNA suppressed estrogenic inhibition of ER{alpha}-positive VSMC proliferation. E4F1 mRNA was abundant in premenopausal female aorta with mild atherosclerotic changes. E4F1 is therefore considered one of the estrogen-responsive genes involving ER{alpha}-mediated inhibition of VSMC proliferation and may play an important role in estrogen-related atheroprotection of human aorta.

Estrogen has been proposed as a cardioprotective agent.1 However it is also true that the significance of hormone replacement therapy has remained controversial because recent randomized controlled trials failed to show protective effects of hormone replacement therapy in reducing the risk of coronary artery disease and instead revealed undesirable side effects such as an increment of breast cancer incidence.2-4 Results of experimental, clinical, and epidemiological studies have, however, also demonstrated that estrogen is predominantly involved in the suppression of development of atherosclerosis.2,5,6 Therefore, it is still very important to examine the detailed mechanisms of estrogenic actions especially in relation to its atheroprotective effects in the human cardiovascular system.

Estrogens have been considered to exert direct anti-atherogenic effects through an initial interaction with estrogen receptor (ER) in vascular smooth muscle cells (VSMCs) in addition to various systemic estrogenic effects. Results of recent studies demonstrated that there are two subtypes of ERs, ER{alpha} and ERß.7,8 The presence of both ER{alpha} and ERß has been also reported in the vascular wall of cardiovascular systems in human and various experimental animals.9 Between both ERs, ER{alpha} has been considered to be important for anti-atherogenic effects of estrogen in the great majority of the cases.9-13 However, the detailed genomic mechanisms of estrogen-ER{alpha} actions in inhibition of VSMC proliferation remain virtually unknown. Especially the target gene (TG) induced by estrogen, ie, estrogen-responsive gene, has not been examined in the human cardiovascular system.

Recently, expression profiling analysis using cDNA microarray technology has been demonstrated to provide very important information as to the elucidation of the scheme of estrogen-signaling and improvement of clinical decisions in ER{alpha}-positive breast cancer.14 A number of investigators also identified several novel estrogen-responsive genes using a cDNA microarray method in other tissues and cancer cells.14,15 However, there has been little information regarding estrogen-responsive genes involving anti-atherogenic effects induced by estrogens in the human cardiovascular system. Therefore, in this study, we first screened the estrogen-responsive gene involving inhibition of VSMC proliferation using a microarray in a cell line derived from ER{alpha}-positive human VSMCs. We then used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate the expression level of this TG mRNA in both dose- and time-dependent manners to further confirm the results of microarray analysis. We also examined whether the TG detected in ER{alpha}-positive VSMCs can be also induced by estrogens in ERß-positive VSMCs. Double-stranded RNAs (dsRNAs) have been recently reported to be remarkably effective at suppressing specific gene expression in various kinds of cells by a pathway involving RNA interference (RNAi) through small interfering RNAs (siRNAs).16-18 Therefore, we used this procedure to confirm whether the gene products derived from the TG detected in microarray analysis were associated with estrogenic inhibition of cell proliferation in ER{alpha}-positive cells.

It then becomes very important to study the expression of the genes detected by microarray analysis in human VSMCs to obtain their clinical relevance. We therefore examined the expression levels of the above TG in VSMCs of human abdominal aorta obtained by autopsy using both quantitative RT-PCR and the in situ hybridization method. We then correlated these findings with the degree of atherosclerosis, sex, ER{alpha} expression levels, and other features of the patients for further characterization of the findings.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Characterization

Two types of human VSMCs, ie, HUVS-112D (derived from human umbilical cord; CRL-2481), and T/G HA-VSMC (derived from human aorta; CRL-1999) were commercially obtained from American Type Culture Collection (Manassas, VA). They were cultured in a 75-cm2 flask with F12-K medium (American Type Culture Collection) containing 5% fetal bovine serum (FBS) at 37°C in a 5% CO2 atmosphere. We first characterized these cell lines using several methods, such as morphological, immunohistochemical, and microarray analyses described below. In addition, we examined whether these cells expressed both ERs, especially ER{alpha}, using quantitative RT-PCR and Western blot analysis in combination with semiquantification, as described below.

Quantitative RT-PCR

Total RNA was extracted from both VSMCs in 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA) followed by a phenol-chloroform phase extraction and isopropanol precipitation. The Superscript Preamplification System RT kit (Life Technologies, Inc., Grand Island, NY) was used in the synthesis and amplification of complementary DNA (cDNA). cDNA was synthesized from total RNA (2 µg) using 25 ng/µL of Oligo (dT)12-18 Primer (Life Technologies Inc., Gaithersburg, MD) on a PTC-200 Peltier thermal cycler DNA engine (MJ Research Inc., Watertown, MA). To test for the presence of genomic DNA contamination, we performed the RT step in the absence of Superscript II RNase H– reverse transcriptase (Life Technologies, Inc.) followed by PCR. RT-PCR products lacking reverse transcriptase in the initial RT step were run on an ethidium bromide-stained 2% agarose gel. No band was observed in these samples (data not shown). The resulting cDNA was used as a template for real-time PCR. Real-time PCR was performed with the Light Cycler System (Roche Diagnostics GmbH, Mannheim, Germany) using the DNA binding dye SYBR Green I (Roche Diagnostics GmbH) for the detection of PCR products. Primers are summarized in Table 1 . As a positive control, T-47D human breast cancer cells were used for ER{alpha} and ERß.19 Negative control experiments lacked cDNA substrate to check for the presence of exogenous contaminant DNA. No amplified products were detected under these conditions. The mRNA levels for both ERs in each VSMC are summarized as a ratio of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and evaluated as a ratio (%) compared with that of each control cDNA, which were synthesized from each PCR products and purified by using the pGEM-T Easy vector. The detailed procedures above were previously described in detail.20 The analyses with real-time PCR were triplicated.


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  		Table 1. Primer Sequences Used in RT-PCR Analysis for VSMC Characterization

 
Western Blot Analysis

The procedures were based on these reported previously.21 The above VSMCs were washed with ice-cold phosphate-buffered saline (PBS) and then lysed in a triple detergent buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate, 100 µg/ml phenylmethyl sulfonyl fluoride, 1 µg/ml aprotin, 1% Nonidet P-40, and 0.5% sodium deoxycholate. A Protein Assay Rapid Kit (Wako, Osaka, Japan) and a SpectraMax 190 microplate reader (Molecular Devices Corp., Sunnyvale, CA) were used to determine the concentration of protein according to the manufacturers’ instructions. A Western blot analysis was performed using 60 µg of each protein. After optimizing the conditions of experiments, 60 µg of protein samples were denatured at 95°C in Tris/glycine/sodium dodecyl sulfate buffer (25 mmol/L Tris, 192 mmol/L glycine, 0.1% sodium dodecyl sulfate, pH 8.3) and electrophoresed at 25-mA constant current through a 10 to 20% polyacrylamide gradient gel (Bio-Rad, Hercules, CA). The proteins in the gel were electrophoretically transferred to a polyvinylidene difluoride membrane (Clear Blot Membrane-P; ATTO Co. Ltd., Tokyo, Japan). Nonspecific binding sites were blocked by immersing the membrane in 5% skim milk (Becton Dickinson and Company, NJ) for 1 hour at room temperature, washed twice in 0.05% Tween 20 and PBS (PBS-T), and then incubated with ER{alpha} polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or control IgG overnight at 4°C. After washing in PBS-T, membranes were incubated for 1 hour with horseradish peroxidase-conjugated anti-mouse IgG (Amersham), and the target protein was detected using the ECL Western blotting detection reagent (Amersham). Equal loading of protein in each lane was confirmed by probing the membrane with anti-human ß-actin monoclonal antibody (Sigma, St. Louis, MO). The relative amounts of ER{alpha} and ß-actin protein levels for each band were standardized to the relative OD units obtained by an Image Gauge system (Fuji Photo Film Co. Ltd., Minamishinagawa, Kanagawa, Japan). In addition, the relative amount of ER{alpha} protein was adjusted by the ß-actin protein level, and then evaluated as a ratio (%) to untreated MCF-7 cell lines.22 The analyses were also triplicated.

GeneChip Microarray Assay

Estrogen Treatment

The two types of VSMC cells described above were seeded in a 75-cm2 flask at an initial concentration of 100,000 cells/flask with F12-K medium (American Type Culture Collection) containing 5% FBS and cultured until a subconfluent state was obtained. The medium was then replaced with FBS-free and phenol red-free medium (modified Eagle’s medium) (Sigma) to arrest the cell growth. After 24 hours, the medium was replaced again with phenol red-free and FBS-free medium in the presence of estrogen (10 nmol/L) or vehicle (0.1% ethanol). After incubation for 8 hours, the cells were subsequently subjected to total RNA extraction for microarray analysis. Total RNA was prepared using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA was further purified using RNeasy columns (Qiagen, Valencia, CA) and treatment with ribonuclease-free deoxyribonuclease I (Qiagen).

Labeling

Isolated total RNA was labeled as described in the Affymetrix (Santa Clara, CA) GeneChip Expression Analysis Technical Manual (revision 3). The labeling is performed in two steps. In the first step, double-stranded cDNAs (ds-cDNAs) were synthesized from total RNA by reverse transcriptase reaction with SuperScript Reverse Transcriptase II (Life Technologies) and an oligo (dT) primer linked to a T7 RNA polymerase-binding site sequence and second strand synthesis reaction with Escherichia coli DNA polymerase, DNA ligase, and RNaseH. In the second step, the resulted ds-cDNAs were used as templates to produce biotin-labeled cRNA (the target) using T7 RNA polymerase in the presence of biotinylated UTP and CTP (Enzo Diagnostics, Farmingdale, NY). The biotin-labeled cRNAs were fragmented in fragmentation buffer and used for the preparation of target solution.

GeneChip Genome Array Hybridization

Fragmented targets are combined with control oligomer (Control Oligo B2, Affymetrix) and control cRNAs (Eukaryotic Hybridization Control kit, Affymetrix) in a hybridization buffer (100 mmol/L MES, 1 mol/L Na+, 20 mmol/L ethylenediaminetetraacetic acid, 0.01% Tween 20). Each target was hybridized to HGU133A using protocols described in the Affymetrix Expression Analysis Technical Manual.

Data Analysis

The procedure was basically based on the previously reported study.23 The fluorescent signals on the slides were scanned by a GeneArray scanner (Affymetrix) and further data processing was performed using Microsoft Excel software (Microsoft, Seattle, WA). Image analysis was performed using Microarray Suite 5.0 software (Affymetrix). In this study, the ratios represented the values changed by 10 nmol/L E2 treatment compared to control values. The values obtained from each experiment were log transformed and normalized so that the median log-transformed ratio equaled zero. To confirm the estrogen-related changes in gene expression obtained from microarray analysis, we independently repeated the same experiment twice and the differences were calculated. To further maintain reproducibility, we also selected for further analysis only those genes in which the difference of expression levels at the same condition in two separate experiments did not exceed more than 2.5-fold in further analysis. The average ratios yielded by these independent experiments were then used to denote the gene expression levels. As the average ratios increased more than twofold by both of duplicated 10 nmol/L E2 treatment were considered to have been up-regulated when compared to control values. In this study, among the genes detected in microarray analysis, we regarded a gene that was up-regulated and was significantly associated with inhibition of cell growth in ER{alpha}-positive cells as the TG in this study. In an analysis of the function of these detected genes, we used the homepage of HUGO Human Gene Nomenclature Committee (http://www.gene.ucl.ac.uk/nomenclature/) for further clarification. We then examined whether there was an estrogen-responsive element (ERE) in the promoter region of TG by analyzing 10,000 bp upstream of the transcription start sites for TG with sequence information of chromosome mapping provided by the above homepage.

Real-Time PCR for TG mRNA

Estrogen Treatment

The ER{alpha}-positive cells were seeded in a 75-cm2 flask at an initial concentration of 100,000 cells/flask with F-12K medium containing 5% FBS and cultured until a subconfluent state was obtained. The medium was then replaced with phenol red-free and FBS-free medium to arrest the cell growth. After 24 hours, the medium was replaced again with FBS-free F12-K medium with E2 (100 pmol/L, 10 nmol/L), E2 (10 nmol/L) with ICI 182780 (1 µmol/L; Tocris, Ballwin, MO), tamoxifen (TAM, Sigma) alone (10 nmol/L), raloxifene (RAL, Sigma) alone (10 nmol/L), or vehicle. In addition, after pretreatment of the cells with inhibitors of RNA transcription, actinomycin D (ACD) (1 µmol/L, Sigma), or protein translation (Sigma), cycloheximide (CHX) (1 µmol/L, ICN Biomedicals Inc.), other two flasks were replaced again with FBS-free medium with E2 (10 nmol/L). After incubation for 8 hours, the cells were subsequently subjected to total RNA extraction for real-time RT-PCR analysis described above for target product mRNA expression. In addition, the cells were also incubated for 24 hours and 48 hours, respectively, with E2 alone (10 nmol/L) or vehicle and also subsequently subjected to total RNA extraction for real-time RT-PCR analysis described above for expression levels of TG product mRNA. The mRNA levels for target product in each VSMC are summarized as a ratio of GAPDH, and evaluated as a ratio (%) compared with that of each control cDNA, which were synthesized from each PCR product and purified by using the pGEM-T Easy vector. The procedures were also previously described in detail.20 As a contrast, we also examined the relative expression levels of TG products mRNA treated with E2 (100 pmol/L, 10 nmol/L) or vehicle for 8 hours, and treated with E2 (10 nmol/L) or vehicle for 24 hours and 48 hours in ERß-positive cells. The analyses with real-time PCR were triplicated by using three flasks per treatment or nontreatment. The sequence information of primers for TG is shown in Table 2 .


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  		Table 2. The Sequence Information of Primers, siRNA, and Probes for in Situ hybridization for Target Gene (E4F1)

 
siRNA Preparation, Transfection, and Cell Count Assay

siRNAs corresponding to the TG mRNAs designed with 5' phosphate, 3' hydroxyl, and two base overhangs on each strand were synthesized and transfected to the ER{alpha}-positive VSMCs. The ER{alpha}-positive cells were seeded in a 25-cm2 flask at an initial concentration of 50,000 cells/flask with F-12K medium containing 5% FBS and cultured until a subconfluent status was achieved. The medium was then replaced with phenol red-free and FBS-free medium to arrest the growth. After 24 hours, transfections of siRNA for endogenous gene targeting were performed with TransMessenger transfection reagent (Qiagen). The TG siRNA (2 µg per flask) was condensed with 4 µl of Enhancer R and formulated with 8 µl of TransMessenger reagent, according to the manufacturer’s instructions. The transfection complex was added directly to the cells; it was replaced with phenol red-free medium containing 5% dextran-coated charcoal-stripped FBS (DCC-FBS) with E2 (10 nmol/L), TAM (10 nmol/L), RAL (10 nmol/L), or vehicle (0.1% ethanol) after 3 hours. After incubation for 48 hours, we then measured the number of cells in each sample as described above. We also examined the number of cells treated with E2 (10 nmol/L) or vehicle (0.1% ethanol) with transfection of negative control siRNA and treated by E2 (10 nmol/L), TAM (10 nmol/L), RAL (10 nmol/L), or vehicle into both ER{alpha}-positive cells. After incubation for 48 hours, the cells were trypsinized and suspended. We then used Cell Counting Kit-8 system (Wako) for measurement of the number of cells in each sample. As a contrast, we also examined the number of cells treated with E2 (10 nmol/L) or vehicle with transfection of TG or negative control siRNA in ERß-positive cells treated by E2. The effective ratio of transfection into the cells was more than 80% by using fluorescein-labeled negative control siRNA before transfection of the TG siRNA (data not shown). The sequence information of siRNA for TG is shown in Table 2 .

Quantitative RT-PCR Analysis for mRNA of the TG Expression in Human Aorta

Human abdominal aortae without hormone therapy were collected at the time of autopsy performed in Tohoku University Hospital (Sendai, Japan) within 2 hours postmortem from 22 patients (6 male, 6 premenopausal female, 10 postmenopausal female; mean, 52.2 ± 5.9 years of age). The Ethics Committee at Tohoku University School of Medicine approved the research protocol for this study. Aortic specimens were tentatively classified into the following five groups as previously described,20 A, B, C, D, and E based on the sex of the deceased patient, degree of atherosclerosis, and status of menstruation (group A: male, normal or mild atherosclerosis, corresponding to group I to III in the AHA classification; group B: male, advanced atherosclerosis, corresponding to group IV to VI in the AHA classification; group C: premenopausal female, normal or mild atherosclerosis; group D: postmenopausal female, advanced atherosclerosis; and group E: postmenopausal female, mild atherosclerosis). The distribution of the cases among these groups is summarized as follows: A, three cases; B, three cases; C, six cases; D, six cases; and E, four cases. The adventitia and fat tissues around the aorta were immediately and carefully removed using clean surgical scissors and forceps at the time of autopsy. After this procedure, these specimens were immediately frozen in liquid nitrogen and stored at –80°C until use and also subsequently subjected to total RNA extraction for real-time RT-PCR analysis described above for expression levels of TG product mRNA and the correlation of ER{alpha} mRNA abundance in those samples. The mRNA levels for target product and ER{alpha} in each sample are summarized as a ratio of GAPDH, and evaluated as a ratio (%) compared with that of each control cDNA, which were synthesized from each PCR product and purified by using the pGEM-T Easy vector, as described above.

In Situ Hybridization Study for the TG mRNA Expression in Human Aorta

Unstained and duplicated, 5-µm-thick, formalin-fixed, paraffin-embedded human aorta sections were mounted onto clear glass slides (Matsunami, Tokyo, Japan) and processed using the RiboMap in situ hybridization kit (Ventana Medical Systems, Tucson, AZ) on the Ventana Discovery (Ventana Medical Systems) automated in situ hybridization instrument. In situ hybridization step protocols after the deparaffinization step were designed based on the standard protocol described in the manufacture’s RiboMap application note. The first fixation step was performed using formalin-based RiboPrep reagent (Ventana Medical Systems) for 30 minutes at 37°C. The reacted sections were then acid-treated using hydrochloride-based RiboClear reagent (Ventana Medical Systems) for 10 minutes at 37°C. The slides were subsequently processed for the protease digestion using ready-to-use a protease 2 reagent. The sections were incubated for hybridization with the anti-sense riboprobe (2 ng/slide) using RiboHybe hybridization buffer (Ventana Medical Systems) for 6 hours at 37°C after a denaturing step for 6 minutes at 70°C. After stringency wash step using 2x RiboWash (equivalent to 0.2x standard saline citrate, Ventana Medical Systems) for 6 minutes at 42°C, the second fixation step was performed using RiboFix reagent for 20 minutes at 37°C followed by incubation of biotin-labeled anti-digoxigenin antibody (Sigma) for 30 minutes at 37°C. After streptavidin-alkaline phosphatase conjugate incubation for 16 minutes at 37°C, the signal was detected automatically using the BlueMap NBT/BCIP substrate kit for 4 hours at 37°C. Finally, the sections were counterstained with Kernechtrot as a marker stain and covered with a glass coverslip. We also used double immunostaining with diaminobenzidine (DAB) for ER{alpha} using a monoclonal antibody (Novocastra Laboratories, Newcastle, UK) and Vector-blue for {alpha}-smooth muscle actin using a monoclonal antibody (DAKO Corp., Carpinteria, CA), respectively, in adjacent tissue sections to further characterize these positive cells in the human aorta of the cases positive for the target mRNAs. We also used a monoclonal antibody against CD31 antigen (DAKO) for endothelial cells and a monoclonal anti-human macrophage antibody (PG-M1, DAKO) for macrophages in adjacent tissue sections for further characterization. In addition, we performed labeling index (LI), a quantitative value that evaluates the number of TG mRNA-positive cells present in VSMCs of the aortic neointima and media.24 After determining the areas of evaluation by simultaneous observation using a multiheaded light microscopy, three authors (T.S., M.S., and H.S.) independently evaluated 100 VSMCs. When interobserver differences were less than 5%, the mean value was determined as the LI. When interobserver differences were greater than 5%, the three aforementioned authors above re-evaluated the discrepant immunostained slides simultaneously using a multiheaded light microscope, after which the mean value was obtained. The sequence information of probes for in situ hybridization for TG is shown in Table 2 .

Statistical Analysis

Values for all results are shown as mean ± SE of means (SEM). For comparisons between two groups, we used one-way analysis of variance followed by unpaired t-test. To examine the correlation of the two factors, we used a correlation coefficient (r) and a regression equation. A P value less than 0.05 was considered significant in this study.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Characterization of Two VSMC Cell Lines

Results are summarized in Figures 1 and 2 . We confirmed that these cells were positive for {alpha}-smooth muscle actin immunoreactivity but both of these cell lines represented relatively dedifferentiated VSMCs on the basis of their morphological features and expression of specific markers such as caldesmon, {alpha}-tropomyosin, and others (data not shown). In RT-PCR analysis, HUVS-112D cells were positive only for ER{alpha} (0.21 ± 0.03%, adjusted by ratios of GAPDH mRNA), and T/G HA-VSMCs were positive only for ERß (0.14 ± 0.01%, adjusted by ratios of GAPDH mRNA) (Figure 1) .



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  		Figure 1. A: Results of real-time RT-PCR analysis for ER{alpha} and ERß in two cultured human VSMC lines (HUVS-112D and T/G HA-VSMC), positive controls, and negative controls. Cell, each type of cultured VSMC; P, positive controls (T-47D breast cancer cell lines); N, negative controls (no cDNAs). T-47D breast cancer cell lines were both positive for ER{alpha} and ERß. Negative controls yielded no bands for both ER{alpha} and ERß. In HUVS-112D cells, only the bands of ER{alpha} were detected, whereas only the bands of ERß were detected in T/G HA-VSMC cells. B: The relative mRNA expression levels for both ERs in two types of VSMC lines. The data for RT-PCR were adjusted by ratios (%) of GAPDH mRNA compared with that of each control cDNA, which were synthesized from each PCR product and purified by using the pGEM-T Easy vector. *, PCR products were not detected and the relative expression levels resulted in zero.

 
In Western blot analysis, only HUVS-112D cells were positive for the full-length ER{alpha} protein (66 kd) (10.4 ± 0.8%, adjusted by ratios of ß-actin protein compared to the ratio of MCF-7 cells) (Figure 2) . In addition, HUVS-112D cells were also positive for the exon 7 deletion variant of ER{alpha} protein (52 kd), which is known to be expressed in both human VSMCs and MCF-7 cells, and known to inhibit transcriptional activation of wild-type ERs with little transcriptional activity by itself (Figure 2) .25,26 On the other hand, MCF-7 cells were also positive for another splicing variant of ER{alpha} protein (46 kd) (Figure 2) .27



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  		Figure 2. A: Western blot analysis of ER{alpha} and ß-actin in HUVS-112D cells, T/G HA-VSMCs, and MCF-7 cells. Total protein was extracted and 60 µg of protein from each cell was loaded. Western blot analysis demonstrated both the full-length ER{alpha} protein (66 kd) in HUVS-112D and MCF-7 cells, but not in T/G HA-VSMCs. In addition, both HUVS-112D and MCF-7 cells were also positive for the exon 7 deletion variant of ER{alpha} protein (52 kd). On the other hand, MCF-7 cells were positive for another splicing variant of ER{alpha} protein (46 kd). B: The relative protein expression levels for the full-length ER{alpha} protein (66 kd) in two types of VSMC lines. The results were evaluated as a ratio (%) compared with that of untreated MCF-7 cells. *, Bands of protein were not detected and the relative expression levels resulted in zero.

 
Gene Chip Microarray Assay

Table 3 shows the 12 genes that demonstrated expression ratios of above 2.0 after 8 hours of duplicated 10 nmol/L E2 treatment of ER{alpha}- and/or ERß-positive cells. Among these genes, E4F transcription factor 1, ie, E4F1 was detected in HUVS-112D cells (ER{alpha}-positive cells), and its expression was associated with the second highest ratios by duplicated 10 nmol/L E2 treatment (2.4-fold) (Table 3) . In addition, among the detected genes, only E4F1 has been reported to inhibit cell growth and cell-cycle progression.28-33 In addition, more than 20 half-EREs in the promoter region of E4F1 were detected in this search (data not shown). Therefore we further examined the features of E4F1 as an estrogen-responsive gene in human VSMCs and whether E4F1 was associated with estrogenic inhibition of ER{alpha}-positive cell proliferation using quantitative RT-PCR, and siRNA transfection assay described above. In addition, we examined the expression levels of E4F1 mRNA in human abdominal aorta using both quantitative RT-PCR and in situ hybridization. In this study, E4F1 was not detected in T/G HA-VSMC cells (ERß-positive cells). In addition, among the genes detected by microarray analysis, the gene associated with inhibition of VSMC proliferation was not demonstrated in ERß-positive VSMCs according to the results of our research.


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  		Table 3. Genes Up-Regulated by Estrogen Treatment for 8 Hours in Cultured VSMCs

 
E4F1 mRNA Expression in VSMCs by Estrogen Treatment

E2 significantly increased E4F1 mRNA levels of ER{alpha}-positive VSMCs compared to controls (131.9 ± 5.9% by E2 100 pmol/L, 267.0 ± 19.7% by E2 10 nmol/L, respectively; P < 0.05) (Figure 3A) . In addition, E2 with CHX also significantly increased those of ER{alpha}-positive VSMCs compared to controls (172.4 ± 20.3%, P < 0.05) (Figure 3A) . However, E2 with ICI 182780, TAM, RAL, and E2 with ACD suppressed expression of these mRNAs (33.4 ± 0.1% by E2 10 nmol/L with ICI 182780 1 µmol/L, 63.5 ± 16.9% by TAM 10 nmol/L, 32.3 ± 1.8% by RAL 10 nmol/L, 48.5 ± 10.6% by E2 10 nmol/L with ACD 1 µmol/L, respectively; P < 0.05) (Figure 3A) . In addition, E4F1 mRNA levels of ER{alpha}-positive VSMCs were the highest at 8 hours after E2 10 nmol/L treatment compared to control (267.0 ± 19.7%) (Figure 3C) . However, none of E2 significantly increased or inhibited E4F1 mRNA levels of ERß-positive VSMCs compared to controls (Figure 3, B and D) . In summary, results of quantitative RT-PCR analyses also demonstrated that E4F1 is one of the genes induced by estrogen via ER{alpha}, but not via ERß, in our cultured human VSMCs.



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  		Figure 3. A: Results of real-time RT-PCR analysis for E4F1 in HUVS-112D cells (ER{alpha}-positive cells) among cells treated with vehicle (control), E2 alone (100 pmol/L, 10 nmol/L), E2 (10 nmol/L) with ICI 182780 (1 µmol/L), E2 (10 nmol/L) with actinomycin D (ACD; 1 µmol/L), E2 (10 nmol/L) with cycloheximide (CHX; 1 µmol/L), tamoxifen (TAM) alone (10 nmol/L), and raloxifene (RAL) alone (10 nmol/L), respectively, after 8 hours. B: Results of real-time RT-PCR analysis for E4F1 in T/G HA-VSMCs (ERß-positive cells) among cells treated with vehicle (control), E2 (100 pmol/L, 10 nmol/L) alone, respectively, after 8 hours. C and D: The relative levels of E4F1 mRNA expression in ER{alpha}-positive cells (C) and ERß-positive cells (D) treated by E2 alone (10 nmol/L) after 8, 24, and 48 hours compared to control (treated by vehicle). *, Significantly increased compared to control; {dagger}, significantly decreased compared to control; P < 0.05.

 
E4F1 siRNA Transfections and Cell Proliferation Assay

We confirmed the down-regulation of E4F1 mRNA levels in the cells by transfection of E4F1 siRNAs using RT-PCR (data not shown). After transfection of negative control siRNA, all E2 and estrogen receptor modulators (SERMs) used in this study significantly inhibited proliferation of ER{alpha}-positive VSMCs compared to controls (20.0 ± 2.2% by E2 10 nmol/L, 28.2 ± 1.0% by TAM 10 nmol/L, and 12.0 ± 0.2% by RAL 10 nmol/L, respectively; P < 0.05) (Figure 4A) . In addition, TAM and RAL significantly inhibited proliferation of ER{alpha}-positive VSMCs compared to controls (30.7 ± 1.6% by TAM 10 nmol/L, and 13.7 ± 1.0% by RAL 10 nmol/L, respectively; P < 0.05) after transfection of E4F1 siRNA (Figure 4A) . However, E2 with transfection of E4F1 siRNA did not suppress the proliferation of ER{alpha}-positive VSMCs (Figure 4A) . In addition, none of the agents examined in this study significantly inhibited the cell proliferation of ERß-positive VSMCs compared to controls by transfection of both E4F1 siRNA and negative control siRNA (Figure 4B) . In summary, siRNA analysis indicated that E4F1 may be associated with inhibition of VSMC proliferation through ER{alpha} not ERß in our cultured VSMCs.



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  		Figure 4. A: The relative levels of cell numbers in HUVS-112D cells (ER{alpha}-positive cells) among cells treated with vehicle (control), estrogen (E2) alone (10 nmol/L), tamoxifen (TAM; 10 nmol/L), raloxifene (RAL; 10 nmol/L), or vehicle (V; 0.1% ethanol) after transfection of E4F1 siRNA (E4F1s) or negative control siRNA (Ns). *, Significantly decreased compared to control; P < 0.05. B: The relative levels of cell numbers in T/G HA-VSMCs (ERß-positive cells) among cells treated with vehicle (control), E2 alone (10 nmol/L), or vehicle (V; 0.1% ethanol) after transfection of E4F1 siRNA (E4F1s) or negative control siRNA (Ns). *, Significantly decreased compared to control; P < 0.05.

 
E4F1 mRNA Expression in Human Aorta

The relative abundance of E4F1 mRNA determined by real-time PCR analysis was significantly higher in the premenopausal female aorta with a mild degree of atherosclerotic changes (group C, 2.22 ± 0.37%) than in the male aorta with a mild degree of atherosclerosis (group A, 0.09 ± 0.05%), with a severe degree of atherosclerosis (group B, 0.31 ± 0.27%), and in the postmenopausal female aorta with a severe degree of atherosclerosis (group D, 0.24 ± 0.12%) (P < 0.05) (Figure 5B) . In addition, there was a significant positive correlation between expression levels of ER{alpha} mRNA and relative abundance of E4F1 mRNA expression in female aorta (y = 0.571 + 1.641 * x, r = 0.65; P < 0.05) (Figure 5C) . In addition, there was a significant inversed correlation between ages of the patients and the abundance of E4F1 mRNA expression in female aorta (y = 2.482 – 0.026 * x, r = –0.575; P < 0.05) (data not shown). However, no significant correlations were detected between relative abundance of E4F1 mRNA and ages and degree of atherosclerosis in male aorta (data not shown). In summary, results of quantitative RT-PCR in human aorta demonstrated that E4F1 was markedly expressed in premenopausal female aorta in an early stage of atherosclerosis with abundant ER{alpha}.



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  		Figure 5. A: Results of real-time RT-PCR analysis for E4F1 in human aortas. Bands for PCR products were detected as specific single bands (167 bp for ER{alpha}, 144 bp for E4F1, and 307 bp for GAPDH). The amplified products were run on a 2% agarose gel stained with ethidium bromide. Representative photographs for these real-time RT-PCR gene products are illustrated. A, Aorta of a 32-year-old man with mild atherosclerotic change; B, Aorta of a 65-year-old man with severe atherosclerotic change; C, aorta of a 38-year-old premenopausal woman with mild atherosclerotic change; D, aorta of a 76-year-old postmenopausal woman with severe atherosclerotic change; E, aorta of a 71-year-old postmenopausal woman with mild atherosclerotic change; P, positive controls; N, negative controls. B: The results for mRNA expression levels for E4F1 (*, P < 0.05). C: Correlation between the levels of mRNA expression for ER{alpha} and E4F1 in 22 samples of human aorta. A significant correlation was detected (y = 0.571 + 1.641x; r = 0.65; P < 0.05).

 
In Situ Hybridization Study for E4F1 mRNA Expression in Human Aorta

Expression of E4F1 mRNA products were markedly present in ER{alpha}-positive VSMCs at neointima of aorta in group C. However, a very low level of expression of both E4F1 mRNA and ER{alpha} immunoreactivity was demonstrated in the nuclei of VSMCs of aorta in other groups. In addition, none of CD31-positive cells or PG-M1-positive cells demonstrated any hybridization signals of E4F1 mRNAs (data not shown). Figure 6 shows representative illustrations of an abdominal aorta specimen obtained from a 38-year-old woman with a mild degree of atherosclerosis (group C). The number of E4F1 mRNA-positive cells in the neointima was significantly higher in the premenopausal female aorta with a mild degree of atherosclerotic changes (group C, 24.0 ± 2.0 LI) than in the male aorta with a mild degree of atherosclerosis (group A, 5.6 ± 0.5 LI), in the male aorta with a severe degree of atherosclerosis (group B, 3.2 ± 0.4 LI), in the postmenopausal aorta with a severe degree of atherosclerosis (group D, 2.4 ± 0.5 LI), and in the postmenopausal aorta with a mild degree of atherosclerosis (group E, 6.2 ± 1.5 LI) (P < 0.05) (Table 4) . However, LI for E4F1 mRNAs in the media was not significantly different among these groups (Table 4) . In summary, results of in situ hybridization study in human aorta demonstrated that E4F1 was markedly expressed in ER{alpha}-positive VSMCs present at neointima of premenopausal female aorta in an early stage of atherosclerosis.



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  		Figure 6. Modified Masson-Goldner’s stains (A), double-immunohistochemical staining photos for {alpha}-smooth muscle actin and ER{alpha} in the neointima (B), and in situ hybridization of E4F1 mRNA in the neointima (C and D) of an abdominal aorta specimen obtained from a 38-year-old premenopausal woman with a mild degree of atherosclerosis (group C). B: Immunopositive cells for ER{alpha} appear brown as a result of diaminobenzidine colorimetric reaction. Immunopositive cells for {alpha}-smooth muscle actin appear blue as a result of Vector-blue colorimetric reaction. Double-immunopositive cells are confirmed. C: Negative control hybridized with sense oligonucleotide probes showed no detectable specific mRNA hybridization signals. Nuclear staining, appearing red, was performed by Kernechtrot. D: E4F1 mRNA hybridization signals, appearing blue, were detected in the marginal region of VSMCs in the neointima. Nuclear staining, appearing red, was performed by Kernechtrot. Ni, neointima; M, media. Original magnifications: x100 (A); x400 (B–D).

 

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  		Table 4. Results of Labeling Index (LI) for Target Gene (E4F1) mRNAs in VSMCs of Human Aorta

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, results of microarray and quantitative RT-PCR analyses all suggest that E4F1 is one of the genes induced by estrogen via ER{alpha}, but not via ERß, in cultured human VSMCs. In addition, siRNA analysis demonstrated that E4F1 might be associated with inhibition of VSMC proliferation through ER{alpha}. Results of quantitative RT-PCR and in situ hybridization studies in human aorta further showed that E4F1 was markedly expressed in ER{alpha}-positive VSMCs present at neointima of premenopausal female aorta in an early stage of atherosclerosis.

In this study, we used duplicated microarray analyses as an initial screening. We then confirmed whether the gene was induced by estrogens or not by triplicated (using three flasks per treatment or nontreatment) quantitative RT-PCR. We then used siRNA assay for further examination of the findings. These findings all demonstrated that results of microarray analysis were consistent with those of RT-PCR, and the gene revealed by microarray analysis was actually involved in estrogenic inhibition of ER{alpha}-positive VSMC proliferation as demonstrated by the cell count assay with siRNA transfection.

E4F1 is well-known as a Krüppel-like family member.28 The growth inhibitory activity of E4F1 has also been demonstrated to be associated with the posttranscriptional elevation of several cell-cycle regulatory proteins, including the CDK inhibitors p21Waf1 and p27Kip1 with reduced cdk2 and cdk4/6 activities, and with the down-regulation of cyclin A and cyclin E gene expression.28-30 E4F1-induced cell-cycle arrest has been also reported to be enhanced by its interaction with the p53 transcription factor and hypophosphorylated pRb.28,32,33 All of these findings above are consistent with the results of previous studies reported, and with inhibitory effects of estrogens on VSMC proliferation.34

Stimulation of responsive genes in response to estrogen is postulated to be mediated by the direct binding in which E2-liganded ER binds directly to a specific sequence called an ERE and interacts directly with co-activator proteins and components of the RNA polymerase II transcription initiation complex which result in enhanced transcription.35 This is also consistent with results of our computer-based search of ERE in the promoter region of E4F1. In addition, quantitative RT-PCR analysis in our present study also demonstrated that ACD suppressed estrogen-induced E4F1 mRNA expression, but CHX did not inhibit its expression. Therefore, these findings all indicated that E4F1 is considered one of the first established responsive genes in ER{alpha}-positive VSMCs. However, it is not known whether these half-EREs are associated with estrogenic actions or not. Therefore, it awaits further investigation for clarification to detect a strict E4F1 promoter region and then to demonstrate whether ER{alpha} is actually binding to each ERE present at upstream region of E4F1 promoter using a chromatin immunoprecipitation assay.36,37

Results of our study in human aorta also demonstrated that levels of E4F1 mRNA expression were significantly higher in VSMCs of neointima of premenopausal female aorta with mild atherosclerotic changes than in those of other groups. This finding therefore suggests that E4F1 is mainly expressed in dedifferentiated/proliferating VSMCs positive for ER{alpha} in neointima of premenopausal female aorta. This finding also demonstrated that the expression of E4F1 may be strongly induced by estrogens via ER{alpha} in VSMCs present at neointima of those aorta, possibly involved in inhibitory actions of VSMC proliferation by estrogens to prevent neointimal formation.38

SERMs are postulated to be an attractive alternative with respect to cardiovascular risk reduction, providing that they exert cardioprotective effects like estrogens. SERMs act as ER antagonists in the breast and uterus, avoiding the harmful effects of estrogens but presumably preserving the beneficial effects of estrogens on bone, lipids, and the cardiovascular system.39-41 In addition, it is known that TAM binds with similar affinity to both ERs, whereas RAL has a higher affinity for ER{alpha}.42,43 Moreover, a previous report demonstrated that TAM has partial agonist activities on anti-atherogenic effects and vascular reactivity through interaction with ER{alpha} but not with ERß in human VSMCs.9 These all may be consistent with results of our present study. It is also postulated that estrogenic effects by SERMs are induced through AP-1 pathway.35 However, several previous reports also documented that pretreatment of SERMs blocked the anti-atherogenic effects exerted by E2 and that they have mixed estrogenic and anti-estrogenic effects on anti-atherogenesis in VSMCs and endothelial cells.44-46 Both TAM and RAL are reported to inhibit the classical ERE pathway described above activated by E2 when these cells are treated together with E2 and SERMs,35 which is also consistent with these above reports.44-46 Results of quantitative RT-PCR and cell proliferation studies also demonstrated that E4F1 was induced by estrogens and suppressed the cell proliferation in ER{alpha}-positive VSMCs, but not by TAM or RAL, which may be partially consistent with results of these above reports. Therefore, SERMs may also be involved in inhibition of ER{alpha}-positive VSMC proliferation not through the above classical pathway induced by E4F1 based on results of our present investigations. These results all suggest the possible involvement of other pathways of estrogenic inhibition of VSMC proliferation. This may be also consistent with a previous report demonstrating that SERMs exert an anti-proliferative effect in VSMCs, at least in part through a p38 cascade whose activation is mediated by ER{alpha} via a nongenomic mechanism.47 However, further investigations are required to determine how these above pathways interact with each other in estrogenic and/or anti-estrogenic effects on VSMCs, and to examine whether there are other possible pathways involved in estrogenic anti-atherogenic processes in human VSMCs in vivo, including an analysis of the difference between E2 and SERMs.

In our study, estrogens demonstrated no inhibitory effects in proliferation of ERß-positive VSMCs. In addition, results of our present study also demonstrated that the inhibitory actions of VSMC proliferation via E4F1 were not detected in these VSMCs, whereas ERß was reported to be predominantly expressed in VSMCs.9 Furthermore, ERß has recently been demonstrated not only to play an essential role in the regulation of vascular function and blood pressure, but also to be associated with several mechanisms of anti-atherogenic effects.48-50 These previous reports of in vitro studies all suggest that E2 was associated with growth inhibitory actions in porcine and/or rat VSMCs predominantly through ERß stimulation.50,51 The signaling pathway mediating growth inhibition via ERß has been also reported to be through reduction of p42/44 and p38 MAPK activity, and/or the cyclic AMP-adenosine pathway.49-52 These signaling pathways may be attributable to the nongenomic action of E2.53,54 However, ERß is well-known as a relatively less potent transactivator than ER{alpha} at low receptor concentrations, such as in our T/G HA-VSMC cell lines, in response to E2.9 This may be one of the reasons to explain the absence of an inhibitory role of estrogenic signals via ERß on VSMC proliferation demonstrated in our present study. ERß is also reported to predominantly increase after injury to the carotid artery of rat, and may be also consistent with promoting inhibitory actions through increment of ERß concentrations.55 On the other hand, previous studies on vascular injury using fully null ER{alpha} knockouts indicated that ER{alpha} is basically and predominantly important for anti-atherogenic effects of estrogen in mice, which is consistent with results of our present study.13 However, according to these several previously published reports, it is difficult to exclude the possibility that which ER is more important for anti-atherogenesis depends on difference of species. Therefore, it still remains unclear which subtype of ER is more important for estrogenic anti-atherogenic effects in human, although our present study demonstrated the above pathway through ER{alpha} and E4F1. In addition, all previous reports have demonstrated several pathways of estrogen-induced anti-atherogenic effects in vitro but not in vivo, and therefore it remains unclear which pathway is most important for ER{alpha}-mediated anti-atherogenic effects in vivo. Further investigations in atherosclerotic models or in human cardiovascular system are required for clarification.

In conclusion, E4F1 is considered one of the estrogen-responsive genes involving ER{alpha}-mediated inhibition of VSMC proliferation and may play important roles in estrogen-related atheroprotection of human aorta.


   Acknowledgements
 
We thank Naomi Kanai for technical assistance.


   Footnotes
 
Address reprint requests to Yasuhiro Nakamura, M.D., Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575 Japan. E-mail: nakamura{at}patholo2.med.tohoku.ac.jp

Supported in part by Health and Labor Sciences Research Grants for Risk Analysis Research on Food and Pharmaceuticals (H13-Seikatsu-013) from the Ministry of Health, Labor, and Welfare of Japan.

Accepted for publication August 25, 2004.


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