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Endocrinology, doi:10.1210/en.2007-0188
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Endocrinology Vol. 148, No. 12 6092-6099
Copyright © 2007 by The Endocrine Society

Mechanism of the Divergent Effects of Estrogen on the Cell Proliferation of Human Umbilical Endothelial Versus Aortic Smooth Muscle Cells

Jun Kawagoe, Masahide Ohmichi, Seiji Tsutsumi, Tsuyoshi Ohta, Kazuhiro Takahashi and Hirohisa Kurachi

Department of Obstetrics and Gynecology (J.K., M.O., S.T., T.O., K.T., H.K.), Yamagata University School of Medicine, Yamagata 990-9585, Japan; and Department of Obstetrics and Gynecology (M.O.), Osaka Medical College, Osaka 569-8686, Japan

Address all correspondence and requests for reprints to: Masahide Ohmich, Department of Obstetrics and Gynecology, Yamagata University School of Medicine, 2-2-2 Iidanishi, Yamagata, Yamagata 990-9585, Japan. E-mail: masa{at}med.id.yamagata-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Diverse estrogen actions are controlled via estrogen receptors (ERs). Mechanisms of action of ERs are modulated by various factors such as ER subtypes, conformation of the ER-ligand complex, and recruitment of coregulator complexes to a target gene promoter. Estrogen exerts divergent actions on vascular cells; namely it increases endothelial cell and inhibits smooth muscle cell growth, resulting in a vasoprotective action. We particularly focused on these divergent effects and examined the mechanisms. The effects of raloxifene, which shows estrogen-like vasoprotective actions, were also examined. To examine the effects of 17ß-estradiol (E2) and raloxifene on human aortic smooth muscle cells (HASMCs) and human umbilical venous endothelial cells (HUVECs), we evaluated the effect of E2 and raloxifene on transcriptional activity, recruitment of the coregulator complex to a target gene promoter, and acetylation of histone of both the IGF-I and COX-2 genes. Treatment with E2 or raloxifene increased both IGF-I and cyclooxygenase (COX)-2 mRNA expression in HUVECs, whereas they attenuated the serum-induced increase of these genes in HASMCs. Treatment by E2 and raloxifene induced recruitment of coactivator complex and histone acetylation at both the IGF-I and COX-2 gene promoter in HUVECs. In contrast, in HASMCs, E2, and raloxifene attenuated the serum-induced recruitment of coactivator complexes and histone acetylation at both the IGF-I and COX-2 gene promoters. Estrogen and raloxifene exert divergent transcriptional regulation on both mRNA expression and the remodeling of IGF-I and COX-2 gene promoters in HUVECs vs. HASMCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MANY EPIDEMIOLOGICAL and basic studies have shown that estrogen increases vasodilatation and inhibits the undesirable response of blood vessels to injury and the development of atherosclerosis (1). Estrogen shows paradoxical effects in vascular endothelial vs. smooth muscle cells. Estrogen accelerates endothelial cell growth in vitro (2) and in vivo (2, 3), whereas it inhibits the migration and proliferation of smooth muscle cells in vitro (4, 5) and in vivo (6). How does estrogen exert these paradoxical actions? To address this question, we refer to the mechanism of the tissue specificity of selective estrogen receptor modulators (SERMs).

Many basic studies about SERMs indicate that the same estrogen receptor (ER)-ligand complex is not recognized similarly in all types of cells. Most of the unique pharmacology of SERMs can be explained by three interactive mechanisms: differential ER expression in a given target tissue, differential ER conformation on ligand binding, and differential expression and binding of coregulator proteins to the ER (7, 8, 9). ER has two isoforms, ER{alpha} and ERß. Each receptor has a distinct action. ER{alpha} is almost always an activator, whereas ERß can inhibit the action of ER a by forming heterodimer with it (8). It was also reported that the agonistic effect of tamoxifen was dependent on a higher concentration of a key coactivator, steroid receptor coactivator (SRC)-1, in the endometrial cells than that in the mammary cells, in which tamoxifen shows an antagonistic effect (9). Thus, variable local concentrations of different coregulator proteins may contribute to the tissue-selective pharmacology of SERMs. This participation of coactivators in the tissue selectivity of SERMs provides a hint for clarifying the paradoxical mechanisms of estrogen between vascular endothelial and smooth muscle cells.

The p160 SRC family includes three homologs, which are SRC-1, SRC-2 [glutamate receptor interacting protein 1 (GRIP-1)], and SRC-3 [amplified in breast cancer 1 (AIB1)] (10). These coactivators interact with ligand-bound nuclear receptors to recruit histone acetyltransferases and methyltransferases to the specific enhancer/promoter regions, which facilitate chromatin remodeling, assembly of general transcription factors, and transcription of target genes (10). In vascular protection, AIB1 has an important role. AIB1 is coexpressed with the ER in the vascular endothelial and smooth muscle cells and may facilitate the estrogen-mediated vasoprotective effects through inhibition of neointimal formation after vessel injury (10, 11). Additionally, AIB1 is involved in IGF-I production (10, 12).

The expression of many substances, such as IGF-I and cyclooxygenase (COX)-2, is correlated with the vasoprotective action of estrogen. IGF-I acts on IGF-I receptors to exert mitogenic activity on both endothelial and smooth muscle cells, and its level is highly correlated with atherosclerosis (13). Estrogen down-regulates IGF-I receptor and IGF-I expression in vascular smooth muscle cells (14), and exerts antimitogenic effects on smooth muscle cells (15, 16). COX-2, expressed in vascular endothelial cells, up-regulates the production of prostacyclin, prostaglandin (PG)-I2 (17, 18). PGI2 exhibits properties with relevance to atheroprotection, inhibiting platelet activation, and vascular smooth muscle contraction and proliferation (19). Some reports showed that estrogen acts on ER{alpha} to activate or up-regulate COX-2 in endothelial cells, resulting in an atheroprotective action (20).

Taken together, these considerations suggest that estrogen has a vasoprotective effect by means of enhancement the proliferation of endothelial cells and inhibition in the proliferation of smooth muscle cells. The mechanism of these effects remains unknown. In the present study, we investigated how these paradoxical actions of estrogen are exerted in terms of IGF-I and COX-2 genes promoter remodeling and cofactor regulation. In addition, we attempted to clarify the effect of raloxifene, because it could also have beneficial effects on endothelial function (37).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Raloxifene analog LY11,718 was a kind gift from Eli Lilly Research Laboratories (Indianapolis, IN). 17ß-Estradiol (E2) and ICI182,780 was obtained from Sigma Chemical Co. (St. Louis, MO). The antinormal rabbit serum (sc-2027), cAMP response element-binding protein (CREB)-binding protein (CBP) (sc-369), SRC-1 (sc-8995), histone deacetylase (HDAC)-2 (sc-7899), nuclear receptor corepressor (NCoR) (sc-1609), silencing mediator of retinoid and thyroid hormone receptors (SMRT) (sc-20778), ER{alpha} (sc-542), ERß (sc-8974), and actin (sc-7210) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antiacetylated histone H4 antibody (06–866) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-GRIP-1 antibody (400070) was obtained from Calbiochem (La Jolla, CA). The anti-AIB1 antibody (AB2831) was obtained from Abcam (Cambridge, UK).

Cell cultures
Human umbilical vein endothelial cells (HUVECs) were isolated from freshly obtained human umbilical cords with type 1A collagenase (1 mg/ml) as described previously (21). HUVECs were cultured at 37 C in endothelial cell growth medium (HuMedia-EG2; Kurabo, Osaka, Japan) containing 2% fetal bovine serum and other growth factor supplements, as supplied by the manufacturer, in a water-saturated atmosphere of 95% O2 and 5% CO2.

Primary cultures of human aortic smooth muscle cells (HASMCs) were obtained from Cascade Biologics (Portland, OR). HASMCs were cultured in vascular smooth muscle cell growth medium (HuMedia-SG2; Kurabo) containing 5% fetal bovine serum and other growth factor supplements, as supplied by the manufacturer, under the same condition as HUVECs.

Unless otherwise indicated, experiments were performed on cells that had previously undergone three or four passages. Before every experiment, these cells were grown to 95% confluence in the respective basal medium and then starved for 24 h in phenol red-free Medium 199 containing 0.4% charcoal-stripped serum (CSS) but lacking any other growth factor supplements to induce quiescence of these cells.

Cell proliferation assay
Cells were plated at a density of 5 x 104 cells per well in six-well plates and allowed to attach overnight. The cells were growth arrested by incubation in phenol red-free Medium 199 with 0.4% CSS for 24 h and were then treated with vehicle, E2, raloxifene, and/or serum (10% CSS) by exchanging the culture medium containing the indicated agent with fresh medium every 48 h for 6 d. A Neubauer chamber was used to count the cell number, and the trypan blue exclusion test was carried out to determine the cell viability. The values shown are the means ± SE of three independent experiments performed in triplicate at three different passages of these cell lines.

RT-PCR analysis of RNA
Total RNA was extracted from cells with Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s instructions. cDNA was prepared from 1 mg of total RNA, primed with random hexamers, and reverse transcribed with Superscript II (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The primers were prepared by Invitrogen and had the following sequences: Cox-2, 5'-TGGGAAGCCTTCTCTAACCTCTCCT-3' (forward) and 5'-CTTTGACTGTGGGAGGATACATCTC-3' (reverse); IGF-I, 5'-TGCTTCCGGAGCTGTGATC-3' (forward) and 5'-AGCTGACTTGGCAGGCTTGA-3'; glyceraldehyde-3-phophate dehydrogenase (GAPDH); 5'-ACCACAGTCCATGCCATCAC-3' (forward) and 5'-TCCACCACCCTGT-TGCTGTA-3' (reverse). Typically, 1-µl aliquots of the reverse-transcribed cDNA were amplified by 25–35 cycles of PCR. Each cycle consisted of denaturation at 94 C for 30 sec, annealing at 55–60 C for 30 sec, and extension at 72 C for 30–45 sec. PCR products were separated by electrophoresis in 8% polyacrylamide gels and visualized by SYBR Green I nucleic acid stain (Cambrex Bioscience Rockland, Inc., Rockland, ME) staining.

Western blot analysis
After treatment the cells were then washed twice with PBS and lysed in ice-cold buffer of 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 µM sodium orthovanadate, 100 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The lysates were centrifuged at 12,000 x g at 4 C for 15 min, and the protein concentrations of the supernatants were determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Equal amounts of proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blocking was done in 10% nonfat milk in 1x Tris-buffered saline. Western blot analyses were performed with various specific primary antibodies. Immunoreacted bands in the immunoblots were visualized with horseradish peroxidase-coupled goat antirabbit or antimouse immunoglobulin by using the enhanced chemiluminescence Western blotting system.

Real-time PCR analysis
The probes and primers used in the real-time PCR analysis were derived from the commercially available TaqMan gene expression assays (Applied Biosystems, Foster City, CA). Assay identifications were Hs99999905_m1 for GAPDH, Hs00153133_m1 for COX-2, and Hs00153126_m1 for IGF-I. Real-time RT-PCR was performed using ABI Prism 7300 (Applied Biosystems) according to the manufacturer’s protocol. All PCRs were performed in triplicate. Reactions were performed by an initial incubation at 50 C for 2 min and at 95 C for 10 min and then cycling at 95 C for 15 sec and 60 C for 1 min for 40 cycles. Quantification was performed by the standard-curve method. A standard curve is generated from a dilution series constructed from a sample. Real-time PCR is performed on both the experimental sample. Relative values for target abundance in each experimental sample were extrapolated from the standard curve. All values for samples were normalized by dividing the concentration of the test gene with the concentration of the GAPDH in the same cDNA sample.

Chromatin immunoprecipitation (ChIP)
We used the technique described for previous study (22). Soluble chromatin was prepared from cells fixed with formaldehyde. The chromatin solution was precleared with a salmon sperm DNA-protein A-agarose 50% slurry (Upstate Biotechnology) for 2 h and then aliquoted and incubated with specific antibodies overnight. Immunoprecipitates were recovered with salmon sperm DNA-protein A-agarose for 2 h, washed with the buffers as described (22), followed by two incubations with freshly made elution buffer [0.5% sodium dodecyl sulfate, 10 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), and 300 mM NaCl]. The formaldehyde cross-linking of protein-DNA complexes were reversed by incubation at 65 C for 4 h. The samples were digested with proteinase K for 1 h at 45 C, and the DNA from the samples was obtained by phenol-chloroform extraction and ethanol precipitation. DNA pellets were then resuspended in 20 µl of sterile water, and 1-µl aliquots were used for the PCRs.

PCR amplifications were performed with the following specific primer pairs designed for each gene promoter: Cox-2, 5'-AAGACATCTGGCGGAAACC-3' (forward) and 5'-ACAATTGGTCGCTAACCGAG-3' (reverse); and IGF-I, 5'-GTCTGCTAACCCTGTCAGAGACAC-3' (forward) and 5'-GGCTCTATCTGCTCTGAATTTAGC-3' (reverse). The PCR products were resolved by 8% PAGE and visualized by SYBR Green I nucleic acid stain (Cambrex Bioscience Rockland) staining under UV illumination.

Small interfering RNA (siRNA) transfection
AIB1 siRNA and nontargeting siRNA were obtained from Dharmacon, Inc. (Dallas, TX). A nontargeting siRNA was used as a control for nonsequence-specific effects of the transfected siRNAs. siRNAs were transfected with DharmaFECT (Dharmacon) into HUVECs and HASMCs following the manufacturer’s protocol.

Statistics
Statistical analysis was performed by Student’s t test, and P < 0.01 was considered significant. Data are expressed as means ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E2 and raloxifene show divergent effects on cell proliferation of HUVECs vs. HASMCs
We evaluated the effects of 10% serum, E2, and raloxifene on the proliferation of HUVECs (Fig. 1AGo) and HASMCs (Fig. 1BGo). At 10 nM, E2 significantly induced the cell proliferation in HUVECs as previously reported (2), whereas 10% serum exerted a more profound proliferative effect. At 10 nM, raloxifene also showed a similar proliferative effect as E2 (Fig. 1AGo). On the other hand, although E2 and raloxifene failed to show a significant effect on HASMCs under quiescent (nonstimulated) conditions, 10 nM E2 and raloxifene inhibited the proliferation of serum-stimulated HASMCs (Fig. 1BGo). These results were consistent with those in previous reports (23, 24).


Figure 1
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  		FIG. 1. E2 and raloxifene show a divergent effect on cell proliferation in HUVECs vs. HASMCs. HUVECs were starved under the 0.4% CSS conditions for 24 h and then cultured with 10 nM E2 (0.4% CSS), 10 nM raloxifene (0.4% CSS), or 10% CSS by exchanging the culture medium every 48 h for 6 d (A). HASMCs were starved under the 0.4% CSS conditions for 24 h and then cultured with 10 nM E2 or 10 nM raloxifene with or without growth stimulation by 10% CSS (B). Western blotting (WB) was performed to confirm the expression of both ER{alpha} and ERß in HUVECs and HASMCs (C). The culture medium was exchanged every 48 h for 6 d. The number of viable cells was counted by the trypan blue exclusion test. The data are shown as the means ± SE (n = 3). Experiments were repeated three times with a consistent result and a representative result is shown. C, Control, a quiescent condition (0.4% CSS); E, E2; R, raloxifene; S, serum (10% CSS) treated.

 
E2 and raloxifene show a divergent effect on IGF-I and COX-2 mRNA expression in HUVECs vs. HASMCs
We first measured the expression of both ER{alpha} and ERß in the different cell type by the Western blotting assay. Both ER subtypes were expressed in both HUVECs and HASMCs. (Fig. 1CGo).

To promote cell growth of HUVECs, estrogen modulates many growth factors, cytokines, and enzymes such as IGF-I (14, 16) and COX-2 (20). We examined whether estrogen and raloxifene regulate the IGF-I and COX-2 mRNA expression in HUVECs. HUVECs were treated with 10 nM E2, 10 nM raloxifene, or 1 mM ICI 182,780 (ER antagonist, a negative control) for 30 min, and semiquantitative RT-PCR and quantitative real-time PCR assays were performed (Fig. 2Go). Treatment of HUVECs with 10 nM E2 (Fig. 2Go, lane 2) or 10 nM raloxifene (lane 3) increased the amount of IGF-I and COX-2 mRNA in 30 min, whereas treatment with ICI 182,780 showed no effect on the expression of either mRNA (lane 4).


Figure 2
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  		FIG. 2. Both E2 and raloxifene increase the amounts of COX-2 and IGF-I mRNA in HUVECs. HUVECs were treated with vehicle (lane 1), 10 nM E2 (lane 2), 10 nM raloxifene (lane 3), or 1 µM ICI 182,780 (lane 4) for 30 min, and then RNA was extracted and RT-PCR assays were performed to detect COX-2 mRNA (panel i) and IGF-I mRNA (panel ii) (A). A representative example of an experiment that was repeated three times is shown. V, Vehicle-treated control; E, E2; R, raloxifene; I, ICI 182,780. COX-2 and IGF-I mRNA were quantified by real-time PCR as described in Materials and Methods. Relative levels of COX-2 (B) and IGF-I (C) mRNAs are shown with the amount of the vehicle group set arbitrarily at 1.0. Values shown represent the mean ± SE from at least three separate experiments. Significant differences vs. the amounts in the vehicle are indicated by asterisks. *, P < 0.01.

 
Although E2 and raloxifene enhanced the cell proliferation of HUVECs, they attenuated the cell proliferation of HASMCs (Fig. 1BGo) (23, 24). Therefore, we examined whether estrogen and raloxifene attenuate the IGF-I and COX-2 mRNA expression in HASMCs. HASMCs were treated with 10 nM E2, 10 nM raloxifene, or 1 mM ICI 182,780 under growth-stimulated (10% CSS) conditions, and the mRNA expression of IGF-I and COX-2 was examined (Fig. 3Go). The amounts of IGF-I and COX-2 mRNAs were markedly increased by 10% CSS (Fig. 3Go, lane 2), compared with 0.4% CSS (lane 1), and treatment with E2 (lane 3), raloxifene (lane 4), or ICI 182,780 (lane 5) decreased the levels of these mRNAs. These results suggested that two ligands of ERs, E2, and raloxifene attenuate the serum-induced expression of IGF-I and COX-2 mRNAs in HASMCs.


Figure 3
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  		FIG. 3. Both E2 and raloxifene attenuate serum-induced mRNA expression of both COX-2 and IGF-I in HASMCs. HASMCs were treated with vehicle (0.1% ethanol, lanes 1 and 2), 10 nM E2 (lanes 3), 10 nM raloxifene (lanes 4) or 1 µM ICI 182,780 (lanes 5) under quiescent (0.4% CSS) or growth-stimulated (10% CSS) conditions for 30 min, and then RNA was extracted and RT-PCR assays were performed to detect COX-2 mRNA (panel i) and IGF-I mRNA (panel ii) (A). A representative example of an experiment that was repeated three times is shown. E, E2; R, raloxifene; I, ICI 182,780; V, vehicle-treated control. COX-2 and IGF-I mRNAs were quantified by real-time PCR as described in Materials and Methods. Relative levels of COX-2 (D) and IGF-I (E) mRNAs are shown with the amount of the vehicle group set arbitrarily at 1.0. Values shown represent the mean ± SE from at least three separate experiments. Significant differences vs. vehicle bands under growth-stimulated conditions (lane 5) are indicated by asterisks. *, P < 0.01.

 
Thus, treatment with E2 or raloxifene caused a divergent effect on IGF-I and COX-2 mRNA expression in HUVECs vs. HASMCs.

Treatment with E2 and raloxifene shows a divergent effect on IGF-I and COX-2 promoter remodeling
Next, we examined the transcriptional responses of IGF-I and COX-2 genes to estrogen and raloxifene in HUVECs and HASMCs. ER-mediated transcriptional activation is associated with the recruitment of coactivators and subsequent histone acetylation. In contrast, antagonist-liganded ER is able to recruit corepressors such as NCoR and SMRT and a subset of HDACs. The association of ER and coregulators with the target gene promoters and the target gene promoter acetylation were evaluated by ChIP assays. HUVECs were treated with 10 nM E2, 10 nM raloxifene, or 1 mM ICI 182,780 for 30 min. PCR amplifications were conducted on a fixed amount of immunoprecipitated DNA, followed by 30 cycles of PCR with specific primer pairs for the human IGF-I or COX-2 promoters.

In HUVECs, treatment with E2 or raloxifene induced the recruitment of ER{alpha} (Fig. 4Go, panel xi) to both COX-2 (lanes 2 and 3) and IGF-I (lanes 6 and 7) promoters. Although treatment with ICI 182,780 (lanes 4 and 8) induced the recruitment of ER{alpha} to both COX-2 (lane 4) and IGF-I (lane 8) promoters, it did not induce the association of coactivator complexes (Fig. 4Go, panels v–viii) to either promoter and did not induce acetylation of histone H4 (Fig. 4Go, panel iii). These results agreed with the current model of mRNA transactivation through ERs. This ligand-stimulated histone H4 acetylation was accompanied by coactivator recruitment. E2 and raloxifene induced the recruitment of coactivators, including CBP (Fig. 4Go, panel v), GRIP-1 (Fig. 4Go, panel vii), and AIB1 (Fig. 4Go, panel viii) to both the COX-2 (lanes 2 and 3) and IGF-I (lanes 6 and 7) promoters. Recruitment of SRC-1 to the COX-2 gene promoter was not observed with E2 and raloxifene, whereas they induced the recruitment of SRC-1 to the IGF-I gene promoter (Fig. 4Go, panel vi). In contrast, treatment with either E2 or raloxifene reduced the association of corepressors, including NCoR (Fig. 4Go, panel ix) and SMRT (Fig. 4Go, panel x) at both the COX-2 (lanes 2 and 3) and IGF-I (lanes 6 and 7) promoters. We also examined the recruitment of ERß on both IGF-I and COX-2 promoters. The modulation of ERß on promoter remodeling were the same as the modulation of ER{alpha} (Fig. 4Go, panel xii, lanes 2, 3, 6, and 7).


Figure 4
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  		FIG. 4. Both E2 and raloxifene induce recruitment of coactivator complex and acetylation of histone H4 in both COX-2 and IGF-I gene promoters and, conversely, reduce corepressor complex and HDAC2 in HUVECs. HUVECs were treated with vehicle (0.1% ethanol, lanes 1 and 5), 10 nM E2 (lanes 2 and 6), 10 nM raloxifene (lanes 3 and 7), or 1 µM ICI 182,780 (lanes 4 and 8). Thirty minutes later, cross-linking of DNA and proteins, and ChIP assays were performed to detect COX-2 (lanes 1–4) and IGF-I (lanes 5–8) gene promoter remodeling and coactivator and corepressor association. A representative example of an experiment that was repeated three times is shown. V, Vehicle-treated control; E, E2; R, raloxifene; I, ICI 182,780; IP, immunoprecipitates.

 
In HASMCs, serum (10% CSS) treatment facilitated the recruitment of ER{alpha} (Fig. 5Go, panel xi) to both the COX-2 (lane 2) and IGF-I (lane 7) promoters. Treatment with E2, raloxifene, or ICI 182,780 reduced the serum-induced recruitment of ER{alpha} (panel xi) to both the COX-2 (lanes 3–5) and IGF-I (lanes 8–10) promoters. HASMCs were treated with the above agents for 30 min under growth-stimulated conditions. Serum treatment modulated the promoter chromatin remodeling via acetylation of histone H4 (Fig. 5Go, panel iii) and decreased the association of HDAC2 (panel iv) and corepressors, including NCoR (panel ix) and SMRT (panel x) to both the COX-2 (lane 2) and IGF-I (lane 7) promoters. However, in HASMCs, treatment with E2 or raloxifene attenuated the serum-induced acetylation of histone H4 (panel iii) and facilitated the recruitment of HDAC2 (panel iv) and corepressors, including NCoR (panel ix) and SMRT (panel x) to both the COX-2 (lanes 3 and 4) and IGF-I promoters (lanes 8 and 9). Among the coactivators studied, only the amount of serum-facilitated AIB1 immunoprecipitates (panel viii) was down-regulated by both E2 and raloxifene at both the COX-2 (lanes 3 and 4) and IGF-I (lanes 8 and 9) promoters. The modulation of ERß on promoter remodeling were same as the modulation of ER{alpha} (panel xii, lanes 2, 3, 7, and 8).


Figure 5
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  		FIG. 5. Both E2 and raloxifene attenuate serum-induced recruitment of coactivator complex and acetylation of histone H4 in both COX-2 and IGF-I gene promoters and, conversely, induce the corepressor complex and HDAC2 in HASMCs. HASMCs were treated with vehicle (0.1% ethanol, lanes 2 and 7), 10 nM E2 (lanes 3 and 8), 10 nM raloxifene (lanes 4 and 9), or 1 µM ICI 182,780 (lanes 5 and 10) under growth-stimulated conditions. Thirty minutes later, cross-linking of DNA and proteins and ChIP assays were performed to detect COX-2 (lanes 1–5) and IGF-I (lanes 6–10) gene promoter remodeling and coactivator and corepressor association. A representative example of an experiment that was repeated three times is shown. C, Control, vehicle treatment under quiescent conditions; E, E2; R, raloxifene; I, ICI 182,780; V, vehicle-treated control; IP, immunoprecipitates.

 
Down-regulation of AIB1 expression with siRNA significantly decreases cell proliferation in HUVECs but not HASMCs
In ChIP assays, it was demonstrated that the SRCs, especially AIB1, were regulated oppositely between in HUVECs and HASMCs by estrogen, raloxifene, and ICI 182,780 (Figs. 4Go and 5Go). To examine the role of AIB1 in cell proliferation of vascular wall, we transfected the specific siRNA into HUVECs and HASMCs and evaluated the effects of 10% serum, E2 and raloxifene on the proliferation of HUVECs (Fig. 6Go, A and B) and HASMCs (Fig. 6Go, C and D). First, to examine the efficiency of siRNA transfection with DharmaFECT into HUVECs and HASMCs, the lamin A/C siRNA were transfected as a positive control, and quantitative real-time PCR assays were performed. The expressions of lamin A/C mRNA were decreased to approximately 30% by transfection of lamin A/C siRNA in both HUVECs and HASMCs (data not shown). Then we examined the expression level of coactivator in cells transfected with either nontargeting siRNA or specific siRNA. Specific siRNA transfection led to a significant suppression in the levels of protein of AIB1 (Fig. 6AGo). Next, we transfected AIB1 siRNA or nontargeting siRNA into HUVECs and HASMCs and evaluated the effects of 10% serum, E2, and raloxifene on the proliferation in these cells. In HUVECs, knockdown of AIB1 resulted in a complete inhibition of growth effect by estrogen, raloxifene, and 10% serum (Fig. 6BGo), whereas in HASMCs, it did not alter the effect by estrogen and raloxifene on the proliferation assays significantly (Fig. 6DGo). Transfection of nontargeting siRNA as a negative control into HUVECs and HASMCs did not significantly suppress the cell growth (Fig. 6Go, B and D).


Figure 6
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  		FIG. 6. AIB1 knockdown suppresses the growth effect of estrogen, raloxifene, and serum in HUVECs. Specific siRNA for AIB1 (siAIB1) or nontargeting siRNA (siNT) were transfected into HUVECs and HASMCs. Decreasing of target protein expression by specific siRNA was confirmed by Western blotting (A). Then HUVECs were starved under the 0.4% CSS conditions for 24 h and cultured with 10 nM E2 (0.4% CSS), 10 nM raloxifene (0.4% CSS), or 10% CSS by exchanging the culture medium every 48 h for 6 d (B and C). HASMCs were starved under the 0.4% CSS conditions for 24 h and then cultured with 10 nM E2 or 10 nM raloxifene with or without growth stimulation by 10% CSS (D and E). The culture medium was exchanged every 48 h for 6 d. The number of viable cells was counted by the trypan blue exclusion test. The data are shown as the means ± SE (n = 3). Experiments were repeated three times and a representative result is shown. WB, Western blotting; C, control, a quiescent condition (0.4% CSS); E, E2; R, raloxifene; S, serum (10% CSS) treated; siAIB1, AIB1 siRNA were transfected; siNT, nontargeting siRNA were transfected.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that not only SERMs but also estrogen showed a divergent transcriptional regulation in vitro between two cell types, venous endothelial and aortic smooth muscle cells. Together these compose one structure, the vessel wall, and are close to each other in vivo. E2 and raloxifene showed a divergent regulation of both IGF-I and COX-2 promoter remodeling and of the expression of these genes’ mRNAs in HUVECs vs. HASMCs.

Proliferation and migration of both vascular endothelial and smooth muscle cells in the arterial wall play an important role in the pathophysiology of atherosclerosis. The present study showed that estrogen facilitated IGF-I expression in HUVECs, whereas it reduced the serum-induced expression of IGF-I in HASMCs. IGF-I is a potent mitogen for both venous endothelial and smooth muscle cells (13). For the vasoprotective action by estrogen, estrogen must decrease IGF-I expression in vascular smooth muscle cells but not in vascular endothelial cells. In this study, both E2 and raloxifene induced the transcriptional activation of IGF-I, acetylation of histone H4, recruitment of coactivator complex to the IGF-I promoter, and a decrease in corepressor complex and HDAC in HUVECs. Conversely, in HASMCs, serum-induced transcriptional activation of IGF-I was inhibited by both E2 and raloxifene. These findings about the regulation of IGF-I transcription are consistent with a large body of evidence that explains the estrogen-induced vasoprotection (16).

This study also showed that estrogen induced COX-2 expression in HUVECs, whereas it reduced the serum-induced expression of COX-2 in HASMCs. Estrogen up-regulates the expression of COX-2, followed by PGI2 production in vascular endothelial cells (12). PGI2 are potent molecules produced by endothelial cells that act synergistically to maintain normal vascular functions, atheroprotection, and inhibition of platelet activation, vascular smooth muscle contraction, and proliferation (19, 20, 25). Besides down-regulating COX-2 in vascular smooth muscle cells, estrogen may reduce the COX-2-dependent proliferative response of smooth muscles by TNF (25). This dual function of estrogen, up-regulation of COX-2 in HUVECs and down-regulation of COX-2 in HASMCs, may result in the atheroprotective action by estrogen.

How do estrogen and raloxifene show a divergent effect on vascular endothelial vs. smooth muscle cells? One of the possible hypotheses may be the differential regulation of coregulators by the signal transduction.

Recently AIB1 was found to associate with the inhibitory-{kappa}B kinase (IKK) (26). IKK phosphorylates AIB1 and promotes nuclear localization of AIB1. In addition, it was reported that AIB1 was able to augment nuclear factor-{kappa}B (NF{kappa}B)-mediated gene transcription (27), suggesting that AIB1 may play an important role in cell survival as well as inflammatory and immune responses. It was also reported that IKK{alpha} phosphorylates SMRT in chromatin, stimulating the exchange of corepressors with the coactivator complexes (28). IKK{alpha}-induced phosphorylation coincides with a loss of chromatin-associated SMRT and with nuclear export of SMRT corepressor, events required for the expression of NF{kappa}B regulated genes, cellular inhibitor of apoptosis-2 and IL-8 (28). IKK is one of the substrates of Akt (29). It was reported that estrogen and raloxifene activate endothelial nitric oxide synthase through a phosphatidylinositol 3-kinase (PI3K)/Akt cascade in HUVECs (15, 30). Thus, both E2- and raloxifene-induced activation of a PI3K/Akt cascade may be involved in the transcriptional activation including recruitment of coactivator complexes and a decrease in corepressor complexes in the target gene promoters in HUVECs.

Not only PI3K/Akt kinase but also the MAPK family may be involved in the coregulator regulation in SRC-1 activation (31) and inhibition of SMRT and NCoR activity (32). Seven consensus MAPK phosphorylation sites were identified in SRC-1, and phosphorylation of these sites by Erk-2 in vitro suggests that a MAPK pathway can affect the SRC-1 activity. Treatment of cells expressing SRC-1 with epidermal growth factor enhanced the ligand-dependent activation of a target reporter gene. These results suggest that phosphorylation may be a regulatory modifier of SRC-1. We have reported that treatment of HASMCs with E2 or raloxifene activates MAPK to induce apoptosis (33). Furthermore, estrogen-induced activation of MAPK inhibits the migration and proliferation of vascular smooth muscle cells (33). In the present study, recruitment of coactivator complexes including SRC-1 to the target gene promoter was not observed in HASMCs, but recruitment by both E2 and raloxifene was observed in HUVECs (Figs. 4Go and 5Go). Whether activation of SRC-1 via MAPK contributes to this promoter trans-activation is unknown, and further study of this issue is needed.

In this study, the most striking observation was the decrease in the recruitment of AIB1 to both the IGF-I and COX-2 promoters by E2 and raloxifene in HASMCs (Fig. 5Go). The result seems to be important for the following reasons. One is that the function of AIB1 is known to be important to prevent neointima formation by estrogen (11). Indeed, knockdown of AIB1 inhibited the growth effect of estrogen, raloxifene, and serum in HUVECs, whereas it did not significantly affect the effect of estrogen and raloxifene on both the serum-induced and quiescent conditions in HASMCs (Fig. 6Go). The results suggest that estrogen- and raloxifene-induced cell proliferation in HUVECs depends on the transcriptional regulation by AIB1, and the divergent regulation of this coactivator, AIB1, in between HUVECs and HASMCs, might be one of the important function on estrogen- and raloxifene-dependent vasoprotection. Second, in HUVECs, recruitment of AIB1 to both the IGF-I and COX-2 promoters was clearly facilitated by E2 and raloxifene (Fig. 4Go). Third, only the recruitment of AIB1 was decreased by E2 and raloxifene treatment in HASMCs. This alteration was unique and consistent among the coactivator complexes between the IGF-I and COX-2 promoters. These observations must be a key to understand the tissue-specific and the vasoprotective actions of estrogen and raloxifene. The mechanism by which these agents regulate the recruitment of AIB1 is not known. Further study is needed to understand how estrogen and raloxifene regulate the divergent effects between HUVECs and HASMCs.

To explain the tissue-specific action of estrogen receptor ligands, the presence of two ER subtypes, ER{alpha} and ERß, is also important. It is widely accepted that IGF-I and COX-2 are induced by estrogen via non-estrogen response element-dependent transcriptional mechanism of ER{alpha} (9, 20). However, whether ER ligands control these transcriptional mechanism via ERß is not well known. Some studies reported that ERß modulates ER{alpha}-mediated transcriptional activation (34, 35, 36). Modulation of ER{alpha}-mediated gene expression by ERß is one of the possibilities to explain the divergent transcriptional regulation of estrogen and raloxifene between endothelial and smooth muscle cells. Although both ER isoforms are expressed in the vascular endothelium and smooth muscle cells (36), the present study could not clarify the interaction of two ER isoforms. Further examination is needed.

In summary, this study is the first to show that both E2 and raloxifene regulate paradoxical dual transcriptional activation in the expression of mRNAs and remodeling of target gene promoters that regulate cell proliferation in HUVECs vs. HASMCs.


   Footnotes
 
This work was supported by Grants-in-Aid for General Science Research 17390445 (to H.K.) and 16591636 (to K.T.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and in part by a Grant-in-Aid for the 21st Century Center of Excellence program from the Japan Society for the Promotion of Science.

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online September 13, 2007

Abbreviations: AIB1, Amplified in breast cancer 1; CBP, cAMP response element-binding protein (CREB)-binding protein; COX, cyclooxygenase; CSS, charcoal-stripped serum; E2, 17ß-estradiol; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phophate dehydrogenase; GRIP, glutamate receptor interacting protein; HASMC, human aortic smooth muscle cell; HDAC, histone deacetylase; HUVEC, human umbilical venous endothelial cell; NCoR, nuclear receptor corepressor; PG, prostaglandin; SERM, selective ER modulator; SMRT, silencing mediator of retinoid and thyroid receptor; SRC, steroid receptor coactivator.

Received February 8, 2007.

Accepted for publication August 31, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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