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. 2008 May 1;149(8):3881–3889. doi: 10.1210/en.2008-0288

Estrogen Receptor-α Overexpression Suppresses 17β-Estradiol-Mediated Vascular Endothelial Growth Factor Expression and Activation of Survival Kinases

Shameena Bake 1, Lijiang Ma 1, Farida Sohrabji 1
PMCID: PMC2488252  PMID: 18450951

Abstract

Estrogen and its receptors influence growth and differentiation by stimulating the production and secretion of growth factors. Our previous studies indicate an increased expression of estrogen receptor (ER)-α and decreased growth factor synthesis in the olfactory bulb of reproductive senescent female rats as compared with young animals. The present study tests the hypothesis that abnormal overexpression of ERα contributes to decreased growth factor synthesis. We developed the HeLa-Tet-On cell line stably transfected with ERα (HTERα) that expresses increasing amounts of ERα with increasing doses of doxycycline (Dox). Increasing doses of Dox had no effect on vascular endothelial growth factor (VEGF) secretion in HTERα cells. However, in the presence of 40 nm 17β-estradiol, VEGF secretion increased in low-dose Dox-exposed HTERα cultures, which was attenuated by the ERα antagonist, 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]1H-pyrazole dihydrochloride. However, at high-dose Dox and, consequently, high ERα levels, estradiol failed to increase VEGF. In the HeLa X6 cell line in which the Tet-On construct is upstream of an unrelated gene (Pitx2A), estradiol failed to induce VEGF at any Dox dose. Furthermore, in the HTERα cell line, estradiol selectively down-regulates phospho-ERK2 and phospho-Akt at high ERα expression. This study clearly demonstrates that the dose of receptor critically mediates estradiol’s ability to regulate growth factors and survival kinases. The present data also support the hypothesis that 17β-estradiol treatment to an ERα overexpressing system, such as the senescent brain, could reverse the normally observed beneficial effect of estrogen.

THE OVARIAN STEROID, estrogen plays a critical role in the development of the central nervous system, cardiovascular system, and skeletal system in addition to its role in reproduction and may play a protective role in neurodegenerative (1,2), cardiovascular (3), and bone diseases (2,4,5). Estrogens effects are mediated by a special class of transcription factors, estrogen receptors (ERs), which belong to a steroid receptor superfamily. The estrogen receptor is a ligand-dependent transcription factor that regulates the expression of estrogen-responsive genes in target tissues. The three major forms of ER are ERα (6), ERβ (7), and truncated ER product as reported in the rat pituitary (8). Although ERα and ERβ share 95% homology in DNA-binding domains, they differ from each other by 45% in their ligand-binding domain, and both are encoded by different genes. The transcriptional efficiency of ERα and ERβ varies with the type of promoter of the target gene (9).

The brain is an important nonreproductive target organ for estrogen, and estrogen regulates the expression of key enzymes, cytoskeletal proteins, and growth factors (summarized in Ref. 10). Many of these processes critically affect neural development (11,12,13,14,15), and several of them, such as its actions on growth factors (10,16,17) and the cholinergic system (4,18,19,20), continue to occur in the adult brain as well. Direct neuroprotective actions of estradiol have been demonstrated by several in vivo and in vitro studies. Estrogen improves learning and memory in rodents (21,22) and cognitive function in aged monkeys (23). 17β-Estradiol decreases the infarct size after occlusion of the middle cerebral artery (2,24) and attenuates the loss of cholinergic function and growth factors after excitotoxic injury (25,26). In many tissues, both in the brain and the periphery, estrogen mediates its cytotrophic effects via regulation of local growth factors and their receptors (27,28).

However, studies from our laboratory show that 17β- estradiol’s neurotrophic effects are not universal. Thus, although acute and chronic 17β-estradiol treatment increased brain-derived neurotrophic factor (BDNF) mRNA and protein expression in the young adult rat brain (16,17,29,30,31,32), 17β-estradiol fails to increase BDNF in the forebrain of older, acyclic (reproductive senescent) female rats (33). Moreover, the immunosuppressive actions of 17β-estradiol demonstrated in inflammation models in young adult females (34,35) was actually reversed in senescent females (35).

Although the mechanism underlying estrogen’s age-dependent actions are not fully understood, one possibility may be the age-dependent alterations in estrogen receptors. For example, the diametrically opposite actions of 17β-estradiol on BDNF expression in the olfactory bulbs of young and senescent females was paralleled by increased expression of ERα in this region in senescent animals compared with young adults (33). Such increases in ERα immunoreactivity have also been observed in other regions of aged female rats, such as the hypothalamus (36).

In the present study, we tested the hypothesis that overexpression of ERα will inhibit the growth-promoting actions of 17β-estradiol. For these studies, a Hela-Tet-On ERα cell line was engineered, in which ER expression was controlled by graded exposure to doxycycline (Dox). Our results indicate that estradiol elevates vascular endothelial growth factor (VEGF) synthesis at low and moderate levels of ERα but fails to increase VEGF at high ERα levels. Furthermore, at high levels of ERα expression, 17β-estradiol treatment suppresses activation of the survival kinases ERK and Akt, suggesting that the dose of ERα is critical in determining growth-promoting actions of estradiol.

Materials and Methods

Construction and analysis of HeLa-ERα cell line

Construction of plasmid.

Human ERα cDNA (kindly provided by Dr. Steve Safe, Texas A&M University) was propagated by cloning into the EcoRI restriction site of the pBluescript SK vector (gift of Dr. Patrick Dunn, Texas A&M University). ERα cDNA was ligated within BamHI and SalI restriction sites, which allows reconstruction of the cDNA into the pTRE2hyg plasmid. The pTRE2hyg plasmid (CLONTECH, Palo Alto, CA) contains the hygromycin-resistance gene localized downstream of tetracycline-response element (TRE) and the cytomegalovirus minimal promoters, enabling activation of ERα expression in the presence of tetracycline or the analogs such as Dox. The pTRE2hygERα construct was verified by restriction digestion and sequencing. The forward and reverse primers for sequencing were: 5′-CGCCTGGAGACGCCATC-3′ and 5′-CCATTCTAAACAACACCCTG-3′, respectively.

Cell culture, gene transfection, and cloning of plasmids

The Hela Tet-On cell line (Invitrogen, Carlsbad, CA) transfected with the plasmid pTet-On and stably expressing reverse tetracycline-controlled transactivator was purchased from CLONTECH. Hela Tet-On cells were maintained at 37 C and 5% CO2 with culture medium including 90% DMEM, 10% Tet-free fetal bovine serum, 4 mm l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml G418. At 90% confluency, the pTRE2hygERα was transfected into Tet-On HeLa cells using the Lipofectamine 2000 reagent (Invitrogen). The cultures were subjected to selection in presence of 200 μg/ml hygromycin (CLONTECH) and 500 μg/ml G418 for more than 2 wk. Clones were then exposed to Dox to assess transfection efficiency. Clones confirmed for stable transfection were passaged and maintained for experimental purposes. Cells were grown in media containing 80% phenol red-free DMEM, 20% gelded horse serum, 4 mm l-glutamine, 500 μg/ml geneticin, and 200 μg/ml hygromycin and were treated with Dox/17β-estradiol (Sigma-Aldrich, St. Louis, MO). HeLa Tet-On cells transfected with an unrelated gene, pTRE-GFP Pitx2A (X6 cell line) used as control cell line, was a kind gift from Dr. Q. Wei (Kansas State University, Manhattan, KS).

RNA extraction and RT-PCR

Total RNA was extracted from the transfected cells using Trizol reagent (Invitrogen) using our previously established methods (37). The concentration of RNA was determined by using Ribogreen RNA quantification kit (Molecular Probes/Invitrogen, Carlsbad, CA). First-strand cDNA synthesis was performed with an Invitrogen first-strand synthesis kit using 2 μg total RNA and oligo deoxythymidine as the primer. The reaction was performed at 50 C for 5 min, 42 C for 60 min, and then heated at 97 C for 5 min. A 2-μl aliquot from each reverse transcription reaction mixture was used for PCR amplification using previously established methods (37). PCRs for VEGF were performed using previously published primers (38). The primer sequences were: forward, 5′-AGGAGGGCAGAATCATCACG-3′, reverse, 5′-CAAGGCCCACAGGGATTTTCT-3′, and the reaction was amplified for 30 cycles, beginning with 95 C for 5 min, and each subsequent cycle at 94 C for 1 min; 55 C for 1 min, and 72 C for 1 min. As an internal control, reverse transcribed product from each treatment condition were also subjected to PCR amplification for a housekeeping gene (cyclophilin) as described elsewhere (37). Primer sequences were: forward, TGG TCA ACC CCA CCG TGT TCT TCG; reverse, TGC CAT CCA GCC ACT CAG TCT TGG. The PCR cycles for cyclophilin are as follows: 95 C for 2 min, 20 cycles of 95 C for 30 sec, 62 C for 1 min, and 72 C for 2 min. PCR products were separated on a 1.5% ethidium bromide-agarose gel and photographed using Molecular Analyst (Bio-Rad, Hercules, CA).

Protein extraction and Western blot analysis

Cultures were rinsed with DMEM, and cell proteins were harvested in lysis buffer [50 mm Tris (pH 7.4), 150 mm NaCl, 10% glycerol, 1 mm EGTA, 1 mm Na-orthovanadate (pH 10), 5 μm ZnCl2, 100 mm NaF, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride in dimethylsulfoxide, 1% Triton X-100] and centrifuged at 18,000 rpm for 15 min. Supernatant was collected and stored at −20 C until further analysis. Protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, IL). Samples (25 μg) were size fractionated on 10% PAGE and transferred to nylon membranes (Millipore, Bedford, MA). Blots were blocked with 1× Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat dry milk. Subsequently blots were incubated with primary (1:200, ERα AB16; Labvision, Fremont, CA) and secondary antibodies (1:5000, goat antirabbit; Upstate, Billerica, MA), subjected to three washes in 1× Tween Tris-buffered saline buffer between the two incubations. The immunosignal was detected to x-ray film (Bio-Rad) using chemiluminescence reagents (Renaissance; NEN Life Science Products, Boston, MA). In the case of ERα-positive bands for Dox 10- and Dox 50-treated cells, the bands were quantified using computer-assisted densitometric analysis (Quantity One; Bio-Rad). Group differences between the band intensity for Dox 10 and Dox 50 groups were analyzed by a t test.

Immunocytochemistry

Cells were rinsed with PBS three times and fixed in 4% paraformaldehyde for 30 min followed by washes in PBS (three times). Cells were blocked for an hour with buffer containing 5% goat serum and sequentially incubated with primary antibodies raised against different epitopes of the C terminus of ERα (1:200; MC20; Santa Cruz Laboratories, Santa Cruz, CA) followed by the appropriate secondary antibodies 1:2000 dilution; Alexafluor 594; Molecular Probes). Labeled cells were coverslipped with Prolong antifade mounting media (Molecular Probes) and images were digitized using the QCapture software (Q-Imaging, Surrey, Canada).

VEGF ELISA

VEGF levels in cell culture media and cell lysates were measured using a quantitative sandwich enzyme immunoassay kit (R&D Systems, Minneapolis, MN) and the manufacturer’s instructions. Briefly, samples and standards were loaded in duplicate onto a 96-well microplate precoated with affinity-purified polyclonal antibody specific for human VEGF. An equal volume of assay diluent was pipetted into all wells containing samples and standards. After 2 h of incubation at room temperature, the unbound antigens were washed five times with wash buffer. Thereafter 100 μl of horseradish peroxidase-conjugated polyclonal antibody was added to each well, and the plate was incubated again for 2 h. The unbound antibody-enzyme was removed by washes (five times). The presence of VEGF was detected by adding 100 μl of substrate for 30 min. The enzyme reaction was stopped by adding 2 n sulfuric acid. The colored reaction product was read at 450 nm in an ELISA plate reader with a correction at 590 nm. The concentration of VEGF present in the samples was interpolated from a linear standard curve using the KC4 software application (BioTek, Winooski, VT).

Phospho-ERK assay

Phosphorylated (p) and total ERK expression was analyzed by Western blots, performed as described above. Briefly, 50 μg of total proteins from each sample were size fractionated on a 10% PAGE gel, electroblotted onto a nylon membrane, and probed with monoclonal antibody for pERK (1:200 dilution; Santa Cruz) and later stripped and reprobed for pan-ERK (1:1000; Santa Cruz). The immunosignal was detected on x-ray film (Bio-Rad) using Chemiluminescence reagent (Renaissance; NEN Life Science Products). X-rays were then digitized and the intensity of the pERK bands were quantified using Quantity-One (Bio-Rad) and normalized to the intensity of the total ERK bands. Data are reported as mean and sem.

Bio-plex phosphoprotein assay

HeLa cell lysates were prepared using the Bio-Plex cell lysis kit (Bio-Rad). Briefly, the cells were first washed with ice-cold wash buffer and then lysed in 100 μl lysis buffer. The lysates were centrifuged at 4500 × g for 20 min at 4 C, and the supernatant was stored at −20 C until further analysis. The total protein concentration was determined using the BCA protein assay kit (Pierce).

The phosphoproteins and total protein targets for ERK and Akt in HeLa cell lysates were determined using Bio-Plex premixed multiplex assay kit (Bio-Rad) according to the manufacturer’s instructions. Briefly, 50 μl of each sample (200 μg/ml concentration) was loaded in duplicate onto a prewet 96-well filter plate containing dye-coupled beads bound to antibodies specific for pERK and pAkt. Equal volume of cell lysis/assay buffer (1:1) was added to replace the cell lysate in blank wells and incubated overnight in a microplate shaker at 300 rpm at room temperature. The following day, unbound proteins were removed by three washes with wash buffer, followed by incubation with biotinylated detection antibodies (25 μl) specific for each target protein for 30 min at room temperature. The plate was vacuum filtered and washed three times with wash buffer and subsequently incubated with streptavidin-phosphatidylethanolamine (50 μl). The plate was rinsed three times with resuspension buffer, and the immunocomplex was suspended in 125 μl of resuspension buffer. The fluorescent signature of the beads was acquired using Bioplex manager software in a Bioplex suspension array system (Luminex 100 system; Bio-Rad). The total proteins for ERK and Akt were assayed similarly in a separate plate. Culture media from these studies was assayed for VEGF.

Statistical analysis

Statistical analysis was performed using a standard statistical package (SPSS, Chicago, IL). For experiments in which VEGF and the survival kinases were measured, a two-way ANOVA was used, coding for Dox dose and 17β-estradiol treatment as the two variables. Planned comparisons (t tests) were used to determine whether specific groups were significantly different from each other. Specific comparisons included Dox 10 vs. Dox 10 + 17β-estradiol and Dox 50 vs. Dox 50 + 17β-estradiol. In experiments in which a high and low Dox dose was used with varying estradiol concentrations and also for ERα antagonist studies, the data were analyzed by a one-way ANOVA with planned comparisons. Bar graphs shown are mean ± sem of a single representative of three independent experiments. Within each experiment, a single group consisted of six replicates. In all cases, group differences were considered significantly different when P ≤ 0.05.

Results

Growth factor regulation by 17β-estradiol under varying levels of ERα expression

Dose response effect of Dox on ERα expression.

Our previous studies indicated that 17β-estradiol treatment dramatically decreased growth factor synthesis in the reproductive senescent brain in which ERα expression was abnormally increased (33). To ascertain the effect of ERα overexpression on 17β-estradiol’s regulation on growth factors, this study used an engineered HeLa cell line and measured VEGF as a prototypic estrogen-regulated growth factor. HeLa cells, transfected with ERα under the control of tetracycline promoter (HTERα), were seeded on 6-well plates and treated, on 60% confluency, with a range (0, 0.1, 1, 10, 20, and 50 μg/ml) of Dox for 24 h. ERα expression was detected by Western blot and immunohistochemistry using a polyclonal antibody raised against the C-terminus epitope of the protein. As shown in Fig. 1Ai, a dose-dependent increase in ERα was observed in response to Dox. This was more prominent at the lower Dox doses (0–1). The intensity of the ER-α band was quantified for Dox 10 and Dox 50 groups which were the salient comparison groups for subsequent experiments. There was a significant increase in ERα expression at Dox 50 compared with Dox 10 (Fig. 1Aii).

Figure 1.

Figure 1

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Dose-dependent ERα expression in HTERα cells. Ai, Western blot analysis of ERα protein expression in ERα-transfected HeLa (HTERα) cells treated with increasing doses of Dox for 24 h. Equal amount of protein (25 μg) was loaded in each lane, and the blot was probed with an ERα-specific antibody (ERα Ab16; Neomarkers, Fremont, CA). Quantitative analysis of the ERα bands at Dox 10 and Dox 50 indicates a small but significant increase in the expression of this receptor at Dox 50, compared with Dox 10 (Aii). B, Whereas no ERα immunoreactivity (using the MC20 antibody) was seen in the Dox 0 treatment (Bi), both cytoplasmic/membrane and nuclear receptors are present once cells are exposed to Dox. Furthermore, there is a dose-dependent increase in ERα-staining intensity between the 10 (Bii) and 50 (Biii) μg/ml Dox dose. No labeling was seen in cells that were not exposed to primary antibody (Biv). Bar, 40 μm. *, P < 0.05.

Whereas the difference between the optical density of the Western blot bands between the Dox 10 and Dox 50 groups is about 8%, the difference in Dox dose-induced ERα expression is more evident in the immunohistochemistry analysis. Whereas no staining was seen in the untreated HTERα cells (Fig. 1Bi), both the nuclear and cytoplasmic forms of ERα were seen at the 10 (Fig. 1Bii) and 50 μg/ml (Fig. 1Biii) Dox dose. Furthermore, whereas some ERα immunopositive cells were seen at the 10 μg/ml Dox (Fig. 1Bii), many more, brightly labeled cells were seen in the 50 μg/ml Dox dose (Fig. 1Biii).

17β-Estradiol-mediated regulation of VEGF as a function of ERα concentration.

Media obtained from HTERα cells treated with Dox in the presence and absence of 17β-estradiol was assayed for VEGF by ELISA. As shown in Fig. 2Ai (black bars), there was a complex regulation of VEGF by 17β-estradiol as a function of increased ERα expression [interaction effect: F(5, 59): 3.17, P < 0.05]. Specifically, at low doses of Dox (hence low doses of ERα), estradiol increased VEGF release (Fig. 2A, white bars). However, at the high dose of Dox and, consequently, high ERα expression, there was a suppression of VEGF levels so that VEGF levels were no different from cells exposed to Dox but without estradiol (Fig. 2A, black bars). To determine whether the difference in media VEGF levels at low and high Dox doses is due to differences in synthesis or secretion, in a separate experiment. VEGF synthesis in protein lysates from high and low Dox-treated cells in the presence and absence of 17β-estradiol was assayed. There was a significant positive correlation between the VEGF levels in the media and lysates in Dox- (both high and low doses) and 17β-estradiol-treated cultures (+0.47, P < 0.05). 17β-Estradiol significantly increased growth factor levels in both cell lysates (20.9%) and media (9.9%) in low Dox-treated cultures and failed to increase VEGF in high-dose Dox exposed cultures.

Figure 2.

Figure 2

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VEGF expression in HeLa cells. HTERα cells were treated with increasing doses of Dox in the presence (40 nm) and absence of 17β-estradiol for 24 h, and VEGF levels were measured by ELISA. A, In the absence of 17β-estradiol, Dox-dependent increase in ERα did not alter the level of VEGF secretion (black bars). 17β-Estradiol treatment significantly increased VEGF in the media at the low Dox doses (0.1–20 μg/ml) (white bars). However, at the high Dox dose (50 μg/ml), 17β-estradiol failed to increase VEGF. *, P < 0.05 (comparisons between vehicle and 17β-estradiol groups at a specific Dox dose). B, HeLa Tet-On cells transfected with an unrelated Dox-induced construct (Pitx2a) was used as a transfection control. Dox treatment did not cause any change in VEGF levels. 17β-Estradiol exposure failed to increase VEGF expression at any of the Dox doses. C, Dose-dependent effect of 17β-estradiol on VEGF release. HTERα cells exposed to low dose of Dox in the presence of increasing estradiol concentration (4, 40, and 400 nm) showed a dose-dependent increase in VEGF, but at high Dox dose, low dose of estradiol reduced VEGF and higher doses (40 and 400 nm) did not alter VEGF. Bars represent the VEGF levels at each dose of estradiol normalized to the baseline VEGF level for each dose of Dox. *, P < 0.05. In all cases, bars represent mean ± sem of a single representative experiment, with n = 6 for each treatment condition.

To establish that the biphasic regulation of VEGF was not a spurious interaction of Dox and 17β-estradiol, we used a control HeLa cell line that was transfected with the same Tet-On construct upstream of an unrelated gene (Pitx2a) (39). This control cell line was cultured under the same experimental conditions and exposed to the same range of Dox in the presence or absence of 17β-estradiol. Dox treatment did not have an effect on VEGF [F(4, 48): 2.45, P ≥ 0.05]. 17β-Estradiol failed to increase VEGF at all Dox doses (Fig. 2B) and in fact appeared to suppress VEGF expression at some doses [F(1, 48): 27.176, P < 0.05]. There was no interaction effect between Dox and 17β-estradiol in the control cell line [F(4, 48): 0.975, P ≥ 0.05].

ERα mediated regulation of VEGF as a function of estradiol concentration

To determine whether VEGF levels are regulated by a certain ratio of estradiol ligand and receptor, the next experiment tested VEGF expression in HTERa cells that were exposed to either low (10 μg/ml) or high (50 μg/ml) Dox with varying concentrations of estradiol (4, 40, and 400 nm). At the low dose of Dox (low ERα), VEGF levels were consistently increased with all estradiol doses (low ERα) [F(3, 22): 3.65, P < 0.05]. Interestingly, there was an overall effect of estradiol doses with the high Dox (high ERα) treatment as well (Fig. 2C) [F(3, 23): 4.89, P < 0.05]. At the lowest dose of estradiol (4 nm), VEGF expression was significantly reduced, compared with the Dox-only group, whereas at the middle and higher doses of estradiol (40 and 400 nm, respectively) VEGF expression was no different from baseline.

Receptor-mediated regulation of VEGF

Estradiol mediated VEGF release in HTERα cell line was further studied in the presence of the ER antagonist, 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]1H-pyrazole dihydrochloride) (MPP; Tocris, Ellisville, MO), which is highly specific for ERα (40). HTERα cells were concurrently exposed to two different low doses of Dox (1 and 10 μg), in which VEGF expression was reliably increased by 17β-estradiol (see Fig. 3A), together with MPP (1 μm) in the presence and absence of 17β-estradiol for 24 h. MPP abolished the estrogen-mediated increase in VEGF at both doses of Dox (Fig. 3A) [F(3, 19): 48.78, P < 0.05], indicating that 17β-estradiol induction of growth factor at the low Dox dose is due to ERα expression.

Figure 3.

Figure 3

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Receptor-mediated regulation of VEGF release. A, Concurrent exposure of HTERα cells to 17β-estradiol and MPP (ERα antagonist) at both the 1 and 10 μg Dox dose completely blocked 17β-estradiol mediated increase in VEGF. *, P < 0.05). Bars represent mean ± sem of a single representative experiment, with n = 6 for each treatment condition. B, RT-PCR indicated that the VEGF primers detected the appropriate size band (342 bp) for the VEGF gene, but there was no variation in the expression of VEGF mRNA as a result of 17β-estradiol treatment or increased Dox doses. Cyclophilin was also amplified from the same samples and used here to indicate that equal amount of RNA was amplified in each case.

Determination of molecular control of VEGF regulation by 17β-estradiol

To determine whether the ERα dose effect on VEGF expression is transcriptional, we analyzed VEGF mRNA expression in HTERα cells exposed to a range of Dox doses, with or without concurrent 17β-estradiol. Cells were harvested for RNA, and VEGF mRNA was determined by semiquantitative RT-PCR. There was no visible change in VEGF mRNA expression as a consequence of Dox or 17β-estradiol treatment (Fig. 3B), suggesting that 17β-estradiol regulation of VEGF protein in the media and in lysates was not due to transcriptional alteration.

17β-Estradiol regulates survival kinases as a function of ERα overexpression

Because VEGF activation and cell survival pathways are highly interdependent, we examined the expression of two major survival signaling kinases, pERK and the phosphorylated serine/threonine protein kinase (pAkt) in HTERα cells. Dox treatment did not alter ERK2 phosphorylation in the absence of 17β-estradiol. In the presence of 17β-estradiol, pERK2 was dramatically decreased in the high Dox dose (50 μg), compared with the low Dox dose (Fig. 4A), indicating that survival pathways are suppressed at high levels of receptor. This pattern of ERK suppression at high levels of Dox was confirmed by the dual laser-based fluorescence assay. Similar to the Western blot analysis, the fluorescent assay also indicated an interaction effect of Dox and estradiol on ERK2 phosphorylation [F(2, 28): 5.593, P < 0.05]. Estradiol significantly decreased phosphorylated ERK2 at high Dox (and therefore high ERα levels) (Fig. 4C). The fluorescent based assay indicated that 17β-estradiol differentially regulates Akt phosphorylation at low and high ERα levels [interaction effect, F(2, 25): 10.072, P < 0.05]. There is a robust increase in Akt phosphorylation with 17β-estradiol at low ERα levels, but phosphorylation of Akt by estradiol was attenuated at high ERα levels. Hence, the inability to increase VEGF expression in the high Dox (consequently high ERα) condition is associated with a critical loss of phosphorylated ERK2 and Akt expression.

Figure 4.

Figure 4

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Regulatory effects of 17β-estradiol on survival kinase expression as a function of ERα concentration. A, Western blot analysis for expression of phosphorylated and total ERK-2 in lysates from HTERα cells treated with increasing amounts of Dox in the presence or absence of 17β-estradiol. Dox treatment alone had no significant effect on ERK2 activation. However, estrogen treatment significantly suppressed pERK2 at the high Dox dose (50 μg/ml). B, Bars represent group means ± sem of the relative OD of pERK2 normalized to total ERK (n = 6 for each treatment condition). *, P < 0.05. C, Dox/17β-estradiol-induced changes in ERK2 phosphorylation were confirmed with the Bio-Plex phosphoprotein assay, indicating again that 17β-estradiol decreased constitutive ERK2 phosphorylation at high ERα expression (n = 6, P < 0.05). D, Dox/17β-estradiol-induced changes in Akt phosphorylation was determined with the Bio-Plex phosphoprotein assay specific for this signaling protein. Bars represent mean ± sem of a single representative experiment, with n = 6 for each treatment condition. The graph represents mean ± sem of phospho-Akt normalized to Akt for each group. 17β-Estradiol increased Akt phosphorylation at low ERα concentrations, but the same dose of estradiol significantly suppressed Akt activation at high ERα expression. *, P < 0.05 (comparisons between vehicle and 17β-estradiol treatment for each Dox dose).

Discussion

The present study tested the hypothesis that ERα overexpression abolishes estradiol’s stimulatory effect on growth factor synthesis. Using the HTERα cell line in which ERα levels can be manipulated by exposure to Dox, we found that 17β-estradiol increased VEGF synthesis at low and moderate levels of ERα but failed to increase VEGF at high ERα levels. The study further confirmed high ER-α expression as the key player in VEGF modulation because of the lack of activational effect of estradiol at physiological and higher (pharmacological) concentrations. These data are reminiscent of our previous results in which high ERα expression in the olfactory bulbs of reproductive senescent females was associated with a loss of estradiol up-regulation on BDNF expression. Unlike the in vivo data, however, which are correlational, the present study directly ties ERα abundance to trophic support. These data also support our hypothesis that there may be a homeostatic dose of ERα necessary for the trophic effect of estrogen (41,42).

Recent studies have revealed a correlation between alterations/increases in ERα expression and disease pathologies in human and experimental animal models. Postmortem brains of Alzheimer’s disease patients show an increased expression of nuclear ERα in the vertical limb of the diagonal band of Broca (43) and also in the hypothalamus of both males and females (44). Similarly, high levels of ERα protein and mRNA have been reported in heart tissue from patients with dilated cardiomyopathy (45). Aging is accompanied by significant increases in ERα expression in the olfactory bulb in senescent animals (30) and hypothalamic neurons of aged female rats (36). Several recent studies also report that the localization of ERα is altered with age. In double-transgenic β-amyloid precursor protein-presenilin mice, nuclear ERα levels decreased in basal forebrain neurons (46). Similarly, in the aged human brain, an increased proportion of nuclear to cytoplasmic ERα was observed in the infundibular nucleus of the hypothalamus (44). Age-related increases in ERα have also been demonstrated in nonneural tissues such as human osteoblast cultures (47) and in the kidneys of female mice (48).

A growing body of evidence also indicates that transfected ERα overexpression potentiates growth inhibition. For example, 17β-estradiol inhibits the growth of ER-expressing HeLa cells (49), reduces the proliferation (50) and metastasis of an ER negative breast cancer cell line (MDA-MB-231) transfected with human ER, and decreases lung metastasis in athymic nude mice injected with ER-expressing cancer cells (51). A recent study demonstrated that overexpression of ERα receptor in the ECV304 and Ishikawa cell line inhibits growth through down-regulation of endothelin-1 and VEGF (52,53). The concept of a dose of ERα provides a plausible explanation for the aforementioned in vivo and in vitro studies, such that low to moderate levels of the receptor stimulate a trophic outcome whereas higher doses predict a more deleterious fate. The present study used VEGF analysis because it is a prototypic estrogen-inducible growth factor and demonstrated that 17β-estradiol increased VEGF levels when cells expressed low levels of ERα and failed to do so when ERα levels were increased. In addition, the present findings also have implications for the therapeutic potential of ER: first, it implies that the transfection of ERα in an ER-negative background can restore the potential for hormone responsiveness, and second, it suggests that the dose of the receptor limits estrogen’s growth-promoting action.

To determine the molecular switch that distinguishes high and low ERα levels in orchestrating 17β-estradiol’s effect on VEGF release, we examined this effect at a transcriptional level and at the protein level. Whereas studies have identified an estrogen response element in the VEGF gene (54) and shown that estrogen up-regulates VEGF mRNA in endometrial carcinoma cell lines (55) and human endometrium (56), no transcriptional changes were observed in the present study. However, protein levels were increased by estradiol in cell lysates of low Dox-exposed cultures, suggesting a posttranscriptional effect of estradiol on VEGF synthesis.

Selected members of the MAPK (ERK) and phosphatidylinositol 3-kinase (PI3-K) signaling (Akt) family play a central role in regulation of VEGF synthesis and release in ERα-positive and -negative cells. 17β-Estradiol treatment to the high ERα-expressing HTERα cells, in which VEGF is suppressed, also suppressed ERK phosphorylation. A precedent for this observation comes from studies in human myeloma cells in which the IL-6-induced increase in VEGF levels are correlated with increased pERK activity, with reduced VEGF secretion in the presence of an ERK inhibitor (57). Collectively, these studies suggest that inhibition of pERK is linearly related to growth factor regulation. The present data are also consistent with studies indicating that 17β-estradiol increases phosphorylation of both ERK1 and ERK2 in ERα-transfected immortalized hippocampal cells (58) and organotypic explants of the rat cerebral cortex (59). Interestingly, exposure to 16α,17-iodo-estradiol, an ERα-specific ligand-attenuated ERK phosphorylation in the same explant cultures (59,60), suggesting that specific activation of this receptor may be inhibitory to this pathway.

VEGF-dependent cell survival is mediated through activation of MAPK (61) and PI3-K signaling pathways (62). Akt, a major effector protein kinase in the PI3-K signaling, is regulated by steroid hormones (63,64), growth factors (65,66,67,68), and their receptors (69,70). MDA-MB-231 cells transfected with nuclear and membrane ERs showed increased Akt and decreased ERK phosphorylation in the presence of 17β-estradiol with slightly different temporal expression patterns between the receptors (41). ERα-mediated VEGF gene expression is shown to be strictly dependent on Akt pathway because in vivo administration of PI3-K inhibitors blocked E2-induced VEGF mRNA in rat uterus (71). In the present study, 17β-estradiol strongly enhanced Akt phosphorylation at low ERα expression and significantly attenuated Akt activation with increased ERα concentration in HTERα cells, and this was paralleled by decreased VEGF levels. Furthermore, the differential response in Akt phosphorylation in HTERα cells reinforces the concept of dose of ERα as a major regulator in estrogen-mediated nongenomic signaling. Although the present study does not allow us to determine whether ERK2 and Akt suppression precedes or follows VEGF regulation, it strongly indicates that 17β-estradiol treatment in the presence of high ERα levels is not cytoprotective.

In the present study, although both nuclear and nonnuclear receptors were increased by Dox treatment (see Fig 1, Bii and Biii), two observations suggest that the abundance of the nonnuclear receptor may be the critical checkpoint for estrogens trophic or nontrophic action. First, 17β-estradiol-mediated increases, and subsequent failure, to induce VEGF (due to receptor dose) was not accompanied by transcriptional regulation of VEGF, and second, at high ERα levels, 17β-estradiol strongly suppressed both ERK2 and Akt phosphorylation. The membrane ER is localized to caveolae or caveolae-like domains that play a crucial role in mediating membrane-associated receptor signaling and the downstream effectors in a variety of cells (72,73). Although the mechanism by which overexpression of this membrane receptor impacts the survival kinases is not clear, one possibility is that ERα overexpression may destabilize the caveolae, causing increased mobilization and endocytosis of this structure. Whereas estrogen signaling and membrane caveolar association has been well demonstrated in nonneuronal cells (72), emerging evidence indicates a similar mechanism of estrogen-induced caveolae-mediated signaling may occur in other cell types as well, notably in neuronal plasma membranes (74,75).

In conclusion, the dose-dependent effect of Dox on 17β-estradiol-mediated VEGF secretion in transfected HeLa cells suggests that the concentration of receptor is a critical checkpoint in estrogens growth-promoting effect. In view of the change in level and pattern of ERα expression in aging and brain pathologies, the present data strongly indicate that the concentration of ERα should be considered an important therapeutic target in estrogen replacement therapy for neural health.

Acknowledgments

The authors thank Dr. Steve Safe (Texas A&M University) for human ERα cDNA; Dr. Patrick Dunn (Texas A&M University) for pBluescript vector; Dr. Qize Wei (Kansas State University, Manhattan, KS) for Pitx2A cell line; and Dr. Wei-Jung Chen (Texas A&M Health Science Center) for advice on statistical analysis.

Footnotes

This work was supported by National Institutes of Health Grants AG 19515 and AG 027684 (to F.S.) and the National Institute of Environmental Health Sciences Grant P30-ES09106, which supported the core facilities used in the study.

Disclosure Summary: The authors have nothing to disclose.

First Published Online May 1, 2008

Abbreviations: BDNF, Brain-derived neurotrophic factor; Dox, doxycycline; ER, estrogen receptor; HTERα, HeLa-Tet-On cell line stably transfected with ERα; MPP, 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]1H-pyrazole dihydrochloride; p, phosphorylated; PI3-K, phosphatidylinositol 3-kinase; TRE, tetracycline-response element; VEGF, vascular endothelial growth factor.

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