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. 2009 Apr 23;150(8):3742–3752. doi: 10.1210/en.2008-1464

Estradiol Increases Guanosine 5′-Triphosphate Cyclohydrolase Expression Via the Nitric Oxide-Mediated Activation of Cyclic Adenosine 5′-Monophosphate Response Element Binding Protein

Xutong Sun 1,a, Sanjiv Kumar 1,a, Jing Tian 1, Stephen M Black 1
PMCID: PMC2717883  PMID: 19389836

Abstract

A number of studies have demonstrated that estradiol can stimulate endothelial nitric oxide synthase expression and activity, resulting in enhanced nitric oxide (NO) generation. However, its effect on the NO synthase cofactor, tetrahydrobiopterin are less clear. Cellular tetrahydrobiopterin levels are regulated, at least in part, by GTP cyclohydrolase 1 (GCH1). Thus, the purpose of this study was to determine the effect of estradiol on GCH1 expression and the regulatory mechanisms in pulmonary arterial endothelial cells. Our data indicate that 17β-estradiol (E2) increases GCH1 transcription in a dose- and time-dependent manner, whereas estrogen receptor antagonism or NO synthase inhibition attenuated E2-stimulated GCH1 expression. Analysis of the GCH1 promoter fragment responsive to E2 revealed the presence of a cAMP response element, and we found that E2 triggers a rapid but transient elevation of phospho-cAMP response element-binding protein (CREB; <1 h) followed by a second sustained rise after 6 h. EMSA analysis revealed an increase in the binding of CREB during E2 treatment and mutation of the cAMP response element in the GCH1 promoter attenuated the E2-mediated increase in transcription. Furthermore, inhibition of the cAMP-dependent kinase, protein kinase A (PKA) completely abolished the E2-stimulated GCH1 promoter activity, whereas the stimulation of cAMP levels with forskolin increased GCH1 promoter activity, indicating the key role of cAMP in regulating GCH1 promoter activity. In conclusion, our results demonstrate that estradiol can modulate GCH1 expression via NO-mediated activation of CREB in pulmonary arterial endothelial cells. These findings provide new insight into the vascular protective effect of estradiol.

Estradiol stimulates GTP cyclohydrolase I transcription in endothelial cells via a signaling pathway involving NO, cAMP, protein kinase A, and CREB.

Nitric oxide (NO) is a labile humoral factor synthesized from the oxidation of the guanidine nitrogen moiety of l-arginine after the activation of NO synthase (NOS) (1). Three isoforms of NOS are known. Constitutive forms are present in endothelial cells (eNOS) and neurons, and a third, inducible isoform is present in macrophages (2,3,4). Tetrahydrobiopterin (BH4) is a cofactor essential for the catalytic activity of all three NOS isoforms (5,6,7). Studies indicate that cellular BH4 levels have important consequences for the structure of NOS. These include the ability of NOS to shift its heme iron to a high spin state; increase arginine binding; and, at least in some NOS isoforms, stabilize the active dimeric form of the enzyme (5,6,7). There are two different metabolic pathways for BH4 generation in cells: the de novo and salvage pathways. The de novo pathway requires three enzymes to generate BH4: GTP cyclohydrolase I (GCH1); 6-pyruvoyl tetrahydrobiopterinsynthase (PTS); and sepiapterin reductase (SR) (8,9). GCH1, is the first, and rate-limiting, enzyme in the pathway, catalyzing the conversion of GTP to 7, 8-dihydrobiopterin triphosphate (8,9). This product is the substrate for PTS, which generates 6-pyruvoyl tetrahydrobiopterin, the substrate for SR to produce BH4 (8,9). The salvage pathway metabolizes sepiapterin and 7,8-dihydrobiopterin to generate BH4.

Accumulated evidence indicates that optimal concentration of BH4 is of fundamental importance for normal function of eNOS in vascular endothelial cells. The exact role of BH4 in the control of eNOS catalytic activity is not completely understood. However, suboptimal concentration of BH4 reduces formation of NO and favors uncoupling of NOS leading to NOS-mediated reduction of oxygen and formation of superoxide anions and hydrogen peroxide rather than NO and is believed to significantly contribute to vascular oxidative stress and endothelial dysfunction (5,7). Recent findings suggest that accelerated BH4 degradation in arteries exposed to oxidative stress may contribute to the pathogenesis of endothelial dysfunction in hypertension, hypercholesterolemia, diabetes, smoking, and ischemia-reperfusion (10,11,12,13,14,15). Increasing evidence suggests that estradiol, which has favorable effects on vasculature, is an important cardiovascular protective molecule. Accumulated studies demonstrate that estradiol can stimulate eNOS activity resulting in the elaboration of NO (reviewed in Ref. 16). However, the molecular mechanisms of estradiol-mediated vascular protection have not been adequately resolved. Thus, the purpose of this study was to determine the effect of estradiol on GCH1 expression in pulmonary arterial endothelial cells (PAECs) and determine the role of NO signaling in this process. Our data indicate that estradiol regulates GCH1 transcription in an NO-dependent manner, and the signaling pathway activated requires both cAMP and protein kinase A (PKA) and leads to increased cAMP response element-binding protein (CREB) binding to the cAMP response element (CRE) site located at position −89 in the GCH1 promoter.

Materials and Methods

Chemicals

17β-Estradiol (E2), 17α-estradiol, and β-estradiol 6-(O-carboxymethyl)oxime-BSA (E2-BSA) were purchased from Sigma (St. Louis, MO). ICI 182,780 and 2-ethyl-2-thiopseudourea (ETU) were obtained from Tocris (Ellisville, MO). H-89 was purchased from Calbiochech (Gibbstown, NJ). Forskolin was obtained from Fisher BioReagents (Fair Lawn, NJ).

Cell culture and treatment

Primary cultures of ovine PAECs were isolated as described previously (10). Briefly, cells were isolated by the explant technique. The heart and lungs were obtained from fetal (138–140 d gestation) lambs after death. These fetal lambs had not undergone previous surgery or study. The main and branching pulmonary arteries were removed and the exterior of the vessel was rinsed with 70% ethanol. The vessel was opened longitudinally and the interior was rinsed with PBS to remove blood. The endothelium was lightly scraped away, placed in medium DME-H16 (with 10% fetal bovine serum and antibiotics), and incubated at 37 C in 21% O2-5% CO2-balance N2. After 5 d, islands of endothelial cells were cloned to ensure purity. Basic fibroblast growth factor (1 ng/ml) was added to the medium every other day. When confluent, the cells were passaged to maintain them in culture or frozen in liquid nitrogen. Endothelial cell identity was confirmed by the typical cobblestone appearance, contact inhibition, specific uptake of acetylated low-density lipoprotein labeled with 1,1′-dioctadectyl-3,3,3′,3′-tetramethylindocarbocyanine Willebrand factor (Dako, Carpinteria, CA). Cells were used before passage 14. At least 24 h before E2 treatment, standard medium was replaced with phenol-red-free DMEM (Invitrogen, Carlsbad, CA) with 10% charcoal-stripped fetal bovine serum (Gemini Bio, West Sacramento, CA) to remove the estrogen-like activity of phenol red and estrogens from the serum. PAECs were then treated with the water-soluble E2 (0–100 nm) for 0–16 h. This dose range was chosen based on two studies in sheep and humans that indicate that circulating estradiol levels vary between about 2–20 nm at parturition and the half-life of estradiol (∼13 h) (17,18). To determine whether the effects were dependent on estrogen receptor (ER), NOS modulation, or cAMP signaling, in certain studies PAECs were pretreated (30 min) with ICI 182,780 (10 μm), the NOS inhibitor ETU (100 μm), or the PKA inhibitor H-89 (5 μm) before E2 exposure. Finally, cAMP levels were induced in PAECs by the addition of the adenylate cyclase activator forskolin (10 μm).

Western blot analysis

Protein extraction was performed following the method of Gueorguiev et al. (22). Cells were collected in lysis buffer containing 125 mm Tris-HCl (pH 6.8), 25% glycerol, 2% sodium dodecyl sulfate, 0.01% bromphenol blue, and 2% β-mercaptoethanol. Equal amounts of proteins from total cell lysates were separated on 4–20% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinyl difluoride membranes. Immunoblotting was carried out using the appropriate antibodies in Tris-base buffered saline with 0.1% Tween 20 and 5% BSA. After washing, the membranes were probed with horseradish peroxidase-conjugated goat antiserum to rabbit or mouse. Reactive bands were visualized using chemiluminescence (Super Signal West Femto; Pierce, Rockford, IL) on a Kodak 440CF image station (New Haven, CT). Bands were quantified using Kodak Image Station software (Kodak 1D 3.6). The GCH1 antiserum was prepared as we have previously described (23); the anti-phospho-CREB antibody, anti-CREB antibody, and anti-ER-β were purchased from Santa Cruz (Santa Cruz, CA), and the anti ER-α was obtained from Upstate (Temecula, CA). Loading was normalized by reprobing the membranes with an antibody specific to β-actin or total CREB, depending on the assay.

GCH1 mRNA analysis

Quantitative RT-PCR using SYBR green I dye for specific detection of double-stranded DNA was used to determine GCH1 mRNA levels in control and estrogen (100 nm for 16 h)-treated PAECs. Briefly, total RNA was extracted from the cells using the RNeasy kit (QIAGEN, Valencia, CA), and 1 μg total RNA was reverse transcribed using QuantiTect reverse transcription kit (QIAGEN, Hilden, Germany) in a total volume of 20 μl. Primers for GCH1, β-actin, PTS, and SR were designed by Primer 3. The sequences were: GCH1 forward, 5′-CAC CAA AGG CTA CCA GGA AA-3′, reverse, 5′-CCG ATA TGG ACC TTT CCA AC-3; PTS forward, 5′-TAT GGA GGA GGC GAT TAT GC-3′, reverse, 5′-CTT TCT GGA GGT TGT CCC AG-3′; SR forward, 5′-AGG AGC TGA AGA GAA AGG GG-3′, reverse, 5′-GAG AGC CCC TAA AAC CAA GG-3′; β-actin forward, 5′-CTC TTC CAG CCT TCC TTC CT-3′, reverse, 5′-GGG CAG TGA TCT CTT TCT GC-3′. Real-time quantitative PCR was conducted on Mx4000 (Stratagene, La Jolla, CA), using 2 μl of RT product, 12.5 μl of QuantiTect SYBR Green PCR master mix (QIAGEN, Hilden) and primers (400 nm) in a total volume of 25 μl. The following thermocycling conditions were employed: 95 C for 10 min, followed by 95 C for 30 sec, 55 C for 60 sec and 72 C 30 sec for 45 cycles. The threshold cycle (Ct) of a serially diluted control sample were plotted to generate a standard curve. Concentration of each sample was calculated by plotting its Ct on the standard curve and then normalized to β-actin (housekeeping gene) mRNA levels.

ER-α and ER-β RNA interference assays

PAECs were transfected with the appropriate small interfering RNA (siRNA) using HiPerFect transfection reagent (QIAGEN) using the manufacturer’s directions. The day before transfection, 2 × 105 cells were seeded in each well of a six-well plate DMEM containing serum and antibiotics. On the day of transfection, the medium was changed to one without antibiotics. For each well 6 μl of a 10-μm siRNA stock (Santa Cruz) of ER-α, ER-β, or the control was diluted into 100 μl of DMEM without serum (to give a final siRNA concentration of 30 nm). To this was added 12 μl of HiPerFect transfection reagent. The solution was vortexed, incubated for 10 min at room temperature, then added drop-wise onto the cells. Validation of ER-α or ER-β silencing in PAECs was carried out by Western blot 24, 48, and 72 h after transfection. For the promoter studies, cells were transfected with the 313GCH1 promoter construct 32 h after siRNA transfection. After a further 24 h, the cells were treated with 17β-estradiol (100 nm) for 16 h before harvest to determine the luciferase activity.

GCH1 promoter analysis

The GCH1 promoter construct, 313 GCH1, has been described previously (24) and was a generous gift from Dr. Gregory Kapatos (Wayne State University, School of Medicine, Detroit, MI). To determine the effect of estrogen on GCH1 transcription, PAECs were cotransfected with 1.6 μg of this plasmid and 0.4 μg of β-galactosidase plasmid (as an internal control to normalize for transfection efficiency) on a 10-cm2 tissue culture plate at 90% confluency with Effectene transfection reagent (QIAGEN, Valencia) according to the manufacturer’s instructions. The next day, the cells were split onto six-well plates and allowed to adhere for overnight. The culture medium was then replaced with phenol red-free DMEM supplemented with 10% charcoal-stripped fetal bovine serum. Twenty-four hours later, cells were pretreated (30 min) or not with ICI 182,780 (10 μm), ETU (100 μm), or 5 μm H-89 (5 μm) and then exposed to 17β-estradiol (100 nm, 16 h). The luciferase activity of 20 μl of protein extract was determined using luciferase assay system (Promega, Madison, WI), and β-galactosidase of 10 μl of protein extract was determined using β-galactosidase enzyme assay system (Promega) as a transfection efficiency control. In addition, a second plasmid was prepared in which a 4-bp mutation was introduced into the CREB consensus sequence within the 313 GCH1 construct. The sequence TGACGCGA at −89 in the human GCH1 promoter (24) was changed to TGcaacGA by site-directed mutagenesis, as described previously (10).

Nuclear extract preparation and EMSA

Nuclear extracts were prepared using the NE-PER nuclear extraction kit (Pierce Biotechnology) as we have described previously (25,26). EMSAs were performed using biotinylated double-stranded oligonucleotides corresponding to the CREB site at −89 of the GCH1 promoter (24). The single-stranded oligonucleotides were biotinylated using biotin 3′ end DNA labeling kit (Pierce Biotechnology) to incorporate 1–3 biotinylated ribonucleotides onto the 3′ end of DNA strands using terminal deoxynucleotidyl transferase and then annealed to make it double stranded. The sequence of the oligonucleotide is CREB: −102 5′-GCG AGG GCC GTG ACG CGA GGC GGG GCC-3′-73 (IDT Technology, Coralville, IA). Binding reactions involved incubating 10 μg of nuclear extract with biotinylated oligonucleotide and 1 μg polydeoxyinosine-deoxycytosine for 20 min at room temperature. The DNA-protein complexes were resolved on a 5% nondenaturing polyacrylamide gel in 1× TBE buffer and then transferred to nylon membrane, and biotinylated oligonucleotide was detected using a LightShift chemiluminescent EMSA kit (Pierce Biotechnology). Competition reactions were carried out using 50- and 100-fold excess of unlabeled oligonucleotides. Supershift experiments were conducted by incubating nuclear extracts for 2 h at room temperature with 4 μg of polyclonal antibodies specific to CREB (catalog no. sc-186X) and phospho-CREB (catalog no. sc-7978) (Santa Cruz Biotechnology) followed by the addition of biotinylated oligonucleotide. The complexes were visualized using an Image station 440 CF (Eastman Kodak).

Measurement of nitrate plus nitrite (NOX) levels

PAECs were treated with 100 nm of E2, 17α-estradiol, or β-estradiol 6-(O-carboxymethyl)oxime-BSA (E2-BSA) for 16 h. After the treatment, NOx levels in the media were quantified using an Apollo 4000 free radical analyzer (World Precision Instruments, Inc., Sarasota, FL).

Statistical analysis

The mean ± sem was calculated for all samples, and significance was determined either by the unpaired t test (for two groups) or ANOVA with Newman-Keuls posttest (for three or more groups). The statistical significance of differences was set at P < 0.05. Statistical analysis was performed using GraphPad Prism version 4.01 for Windows (GraphPad Software, San Diego, CA).

Results

Estradiol increases GCH1 expression in pulmonary arterial endothelial cells

To examine the effect of estradiol on GCH1 protein expression, we initially carried out a dose-response analysis with E2 for 16 h. Western blot analysis identified a dose-dependent increase in GCH1 expression in response to E2 (Fig. 1A). To determine whether the effect detected was a physiological rather than pharmacological effect, we tested the effect of 17α-estradiol (an inactive isomer) on GCH1 expression. Our data indicate that 17α-estradiol does not increases GCH1 expression (Fig. 1A). We next examined the time-course induction of GCH1 expression using 100 nm E2. We found that GCH1 protein expression significantly increased after 6 h of treatment, and this peaked after 16 h treatment (Fig. 1B). Next, we confirmed the requirement for ER activation. We found that the ER antagonist, ICI 182,780 (10 μm) abolished E2-induced GCH1 expression (Fig. 1C). Furthermore, treatment with E2 (100 nm, 16 h) significantly increased the mRNA levels of GCH1 (Fig. 1D), PTS (Fig. 1E), and SR (Fig. 1F).

Figure 1.

Figure 1

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Effect of E2 on GCH1 expression in PAECs. A, Cells were treated with E2 (0–100 nm) or 17α-estradiol (100 nm) for 16 h. Whole-cell extracts (20 μg) were then prepared and immunoblots were performed using an antibody raised against GCH1. Loading was normalized by reprobing the membranes with an antibody specific to β-actin. E2 exposure produced a dose-dependent increase in GCH1 protein levels. However, the bioinactive isoform, 17α-estradiol had no effect on GCH1 protein levels. Values are expressed as mean ± sem, n = 4. *, P < 0.05 vs. untreated; †, P < 0.05 vs. previous dose. B, PAECs were incubated with E2 (100 nm) for 0, 1, 3, 6, 12, or 16 h. Whole-cell extracts (20 μg) were then prepared and immunoblots were performed using an antibody raised against GCH1. Loading was normalized by reprobing the membranes with an antibody specific to β-actin. E2 causes a time-dependent increase in GCH1 protein levels. Values are expressed as mean ± sem, n = 4. *, P < 0.05 vs. untreated; †, P < 0.05 vs. previous time point. C, PAECs were pretreated with the ER antagonist, ICI 182,780 (10 μm, 30 min), followed by treatment with E2 (100 nm, 16 h). Whole-cell extracts (20 μg) were then prepared and immunoblots were performed using an antibody raised against GCH1. Loading was normalized by reprobing the membranes with an antibody specific to β-actin. ER antagonism attenuates the E2-mediated increase in GCH1 protein levels. Values are expressed as mean ± sem, n = 4. *, P < 0.05 vs. untreated; †, P < 0.05 vs. E2 alone. D–F, PAECs were treated with E2 (100 nm, for 16 h), and then total RNA was isolated and analyzed by real-time RT-PCR. E2 increases GCH1 (D), PTS (E), and SR (F) mRNA levels. Values are expressed as mean ± sem, n = 6–9. *, P < 0.05 vs. untreated.

Estradiol stimulates GCH1 expression through membrane-initiated ER signaling

To investigate whether the effect of estradiol on GCH1 was transcriptional and the role of membrane initiated ER signaling, we next examined the effect of E2, E2-BSA (E2 covalently linked to membrane impermeable BSA), or 17α-estradiol (100 nm, 16 h) on a human GCH1 promoter linked to luciferase (313GCH1). Both E2 and E2-BSA significantly increased GCH1 promoter activity (Fig. 2A), demonstrating that increase in GCH1 expression by estradiol was both membrane initiated and transcriptional. To elucidate which ER was involved in the signaling cascade, we next used specific siRNAs against ER-α and ER-β. ER-α siRNA transfection significantly reduced ER-α protein levels (∼50%) without altering ER-β protein levels (Fig. 2, B–D). Similarly, with ER-β siRNA transfection, ER-β protein levels were significantly reduced (∼50%) without any change in ER-α protein levels (Fig. 2, F–H). Surprisingly, we did not observe any change in E2-induced GCH1 promoter activity when an siRNA was used against ER-α or ER-β alone (Fig. 2, E and I), suggesting that the estradiol signal can be transduced through either receptor. To test this, we transfected cells with siRNAs against ER-α and ER-β in tandem. This resulted in significant reduction in the protein levels of both receptors (Fig. 2, J–L) and also resulted in a significant attenuation in the E2-mediated increase in GCH1 promoter activity (Fig. 2M). Together these results demonstrate that both ER-α and ER-β are involved in estradiol-induced GCH1 expression, and either ER-α or ER-β is sufficient to mediate signaling to GCH1 promoter.

Figure 2.

Figure 2

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E2 stimulates GCH1 promoter activity via membrane initiated signaling through both ER-α and ER-β. A, PAECs were transfected with the 313GCH1 promoter-luciferase construct. After treatment (100 nm, 16 h) with E2, E2-BSA, or 17α-estradiol, the luciferase activities were measured. Treatment with E2 or E2-BSA, but not 17α-estradiol, significantly increased GCH1 promoter activity. Values are expressed as mean ± sem, n = 6. *, P < 0.05 vs. untreated. B–D, PAECs were transfected with an siRNA for ER-α or a scrambled siRNA (as a control). Whole-cell extracts (20 μg) were then prepared after 72 h and subjected to Western blot analysis. An approximate 50% reduction in ER-α expression was observed at 72 h after ER-α siRNA transfection (B and C), whereas ER-β protein levels were unchanged (B and D). Values are expressed as mean ± sem, n = 3. *, P < 0.05 vs. scrambled siRNA. E, PAECs were transfected with siER-α or scrambled siRNA (as a control) along with the 313GCH1 promoter-luciferase construct as described in Materials and Methods. After 72 h, the cells were treated with E2 (100 nm, 16 h), and luciferase activity was measured. E2 increased GCH1 promoter activity, even though ER-α protein levels were decreased. Values are expressed as mean ± sem, n = 6. *, P < 0.05 vs. scrambled siRNA. F–H, PAECs were transfected with an siRNA for ER-β or a scrambled siRNA (as a control). Whole-cell extracts (20 μg) were then prepared after 72 h and subjected to Western blot analysis. An approximately 50% reduction in ER-β expression was observed at 72 h after ER-β siRNA transfection (F and G), whereas ER-α protein levels were unchanged (F and H). Values are expressed as mean ± sem, n = 3. *, P < 0.05 vs. scrambled siRNA. I, PAECs were transfected with siER-β or a scrambled siRNA (as a control) along with the 313GCH1 promoter-luciferase construct as described in Materials and Methods. After 72 h, the cells were treated with E2 (100 nm, 16 h), and luciferase activity was measured. E2 increased GCH1 promoter activity, even though ER-β protein levels were decreased. Values are expressed as mean ± sem, n = 6. *, P < 0.05 vs. scrambled siRNA. J–L, PAECs were transfected with siRNAs for both ER-α and ER-β or a scrambled siRNA (as a control). After 72 h, whole-cell extracts (20 μg) were prepared and subjected to Western blot analysis. A 50% reduction in both ER-α (J and K) and ER-β (J and L) expression was obtained. Values are expressed as mean ± sem, n = 3. *, P < 0.05 vs. scrambled siRNA. M, PAECs were transfected with siRNAs for both ER-α and ER-β or a scrambled siRNA (as a control) along with the 313GCH1 promoter-luciferase construct as described in Materials and Methods. After 72 h, the cells were treated with E2 (100 nm, 16 h) and then the luciferase activity was measured. E2 failed to increase GCH1 promoter activity when ER-α and ER-β expression were both decreased. Values are expressed as mean ± sem, n = 6. *, P < 0.05 vs. scrambled siRNA.

NOS inhibition attenuates the estradiol-induced increase in GCH1 expression in pulmonary arterial endothelial cells

Previous studies have shown that estradiol can induce NO production in cultured cells, by increasing eNOS expression and activity. Thus, we next verified a similar effect was occurring in our PAECs. We found an increase in NO production in PAECs exposed to E2 and E2-BSA but not in those exposed to E2 (Fig. 3A), indicating that E2 increases NO production through membrane-initiated signaling. To establish a link between NO signaling and GCH1 expression, PAECs were exposed to the NOS inhibitor ETU (100 μm) for 30 min before E2 (100 nm, 16 h) exposure. We found that NOS inhibition abrogated the E2-mediated increase in GCH1 expression (Fig. 3B).

Figure 3.

Figure 3

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Estradiol increases NOx production through eNOS in PAECs. A, PAECs were treated for 16 with 100 nm of E2, E2-BSA, or 17α-estradiol. NOx production was then measured. E2 or E2-BSA, but not 17α-estradiol, significantly increased NOx production. Values are expressed as mean ± sem, n = 6. *, P < 0.05 vs. untreated. B, PAECs were pretreated with the NOS inhibitor, ETU (100 μm, 30 min) followed by treatment with E2 (100 nm, 16 h). Whole-cell extracts (20 μg) were then prepared and immunoblots were performed using an antibody raised against GCH1. Loading was normalized by reprobing the membranes with an antibody specific to β-actin. NOS inhibition attenuates the E2-mediated increase in GCH1 protein levels. Values are expressed as mean ± sem, n = 6. *, P < 0.05 vs. untreated; †, P < 0.05 vs. E2 alone.

Estradiol induces the phosphorylation of CREB in PAECs

The 313GCH1 construct contains a CRE at position −89. Thus, we next addressed whether estradiol-mediated activation of CREB could be involved in the up-regulation of the GCH1 promoter. Initially, we evaluated the effect of E2 on the expression and phosphorylation of CREB. Our results demonstrate that although E2 did not alter levels of total CREB protein (Fig. 4A), E2 increased the phosphorylation of CREB within 15 min, followed by decline to baseline and a second sustained rise after 6 h (Fig. 4A). In addition, we found that ER antagonism or NOS inhibition attenuated the E2-induced phosphorylation of CREB (Fig. 4B). To determine the potential role of CREB on the regulation of GCH1 promoter activity by E2, we transfected PAECs with a promoter construct identical with the 313GCH1 except for a 4-bp mutation in the wild-type CRE sequence at −89 (from TGACGTCA to TGcaacCA). Using this construct, we were unable to detect any increase in promoter activity by E2 (Fig. 4C).

Figure 4.

Figure 4

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Estradiol regulates GCH1 expression through the PKA-CREB pathway in PAECs. A, PAECs were treated with E2 (100 nm) for 0, 15 min, 1 h, 3 h, 6 h, 12 h, or 16 h. Whole-cell extracts (20 μg) were then prepared and immunoblots were performed using antibody raised against phospho-CREB (pCREB). Loading was normalized by reprobing the membranes with an antibody specific to CREB. Estradiol increases phospho-CREB levels in PAECs. Values are expressed as mean ± sem, n = 3. *, P < 0.05 vs. untreated. B, PAECs were pretreated with ICI 182,780 (10 μm) or ETU (100 μm) for 30 min, followed by treatment with E2 (100 nm, 6 h). Whole-cell extracts (20 μg) were then prepared and immunoblots were performed using antibody raised against phospho-CREB (pCREB). Loading was normalized by reprobing the membranes with an antibody specific to CREB. Both ER antagonism and NOS inhibition attenuate the estradiol-induced phosphorylation of CREB. Values are expressed as mean ± sem, n = 4. *, P < 0.05 vs. untreated; †, P < 0.05 vs. E2 alone. C, PAECs were transfected with 313GCH1 or 313mutGCH1 promoter constructs and then pretreated with ICI 182,780 (10 μm) or ETU (100 μm) for 30 min, followed by treatment with E2 (100 nm, 16 h) and the luciferase activities determined. The estradiol-mediated increase in 313GCH1 promoter activity is inhibited by either ER antagonism or NOS inhibition. Values are expressed as mean ± sem, n = 6. *, P < 0.05 vs. untreated. The 313mutGCH1 promoter contains a 4-bp mutation in the CRE consensus sequence at −89. Mutation of the CRE attenuates the estradiol-mediated increase in GCH1 promoter activity. D, PAECs were transfected with the 313GCH1 or 313mutGCH1 promoter constructs. They were then treated with forskolin (10 μm, 16 h) and the luciferase activities determined. Forskolin significantly increased the activity of the 313GCH1 promoter construct. However, mutation of the CRE element attenuated the forskolin-mediated increase in GCH1 promoter activity. Values are expressed as mean ± sem, n = 6. *, P < 0.05 vs. untreated; †, P < 0.05 vs. 313GCH1 + forskolin. E, PAECs were transfected with the 313GCH1 or 313mutGCH1 promoter constructs. They were then pretreated the PKA inhibitor, H-89 (5 μm, 30 min) followed by E2 (100 nm, 16 h). H-89 completely blocks the estradiol-mediated induction in 313GCH1 promoter activity. Again the mutated CRE is unaffected by estradiol or PKA inhibition. Values are expressed as mean ± sem, n = 6. *, P < 0.05 vs. untreated; †, P < 0.05 vs. estradiol alone.

Modulating cAMP-PKA signaling alters GCH1 promoter activity in pulmonary arterial endothelial cells

PAECs were transfected with 313GCH1 or 313mut GCH1 promoter constructs. After 24 h, cAMP signaling was then enhanced using the adenylate cyclase activator, forskolin (100 μm, 6 h), or inhibited by pretreatment with the PKA inhibitor, H-89 (5 μm, 30 min). PKA inhibition was then followed by E2 exposure (100 nm, 16 h). We found that forskolin alone increased the activity in the wild-type, 313GCH1 promoter construct but not the construct in which the CRE had been mutated, 313mutGCH1 (Fig. 4 D). Conversely, PKA inhibition prevented the E2-mediated increase in wild-type 313GCH1 promoter activity but had no effect on the activity of 313mutGCH1 (Fig. 4E).

Estradiol increases CREB binding to the GCH1 CRE

We next examined the effect of estradiol on binding of CREB at −89 of the GCH1 promoter. We performed EMSA using nuclear extracts prepared from PAECs treated with E2 (100 nm, 16 h). Compared with control extracts, we observed a significant increase in CREB binding (Fig. 5, A and B). The specificity of the binding was demonstrated by the fact that the CREB complex was competed out with 50–100 times excess unlabeled CREB oligonucleotide (Fig. 5C). In addition, we found that ER antagonism or NOS inhibition attenuated the E2-induced increase in CREB binding (Fig. 5, A and B). The specificity of CREB-DNA binding was also confirmed using supershift analysis using antibodies against CREB and phospho-CREB. The presence of a higher migrating supershifted band after incubation with anti-CREB and antiphospho-CREB polyclonal antibodies provides evidence that increased signal in estradiol-treated cells is due to CREB in the binding complex (Fig. 5D).

Figure 5.

Figure 5

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Effect of estradiol on CREB binding to the CRE of the human GCH1 promoter. A and B, PAECs were pretreated with ICI 182,780 (10 μm) or ETU (100 μm) for 30 min, followed by treatment with E2 (100 nm, 16 h) and nuclear extracts prepared. Nuclear extracts (10 μg) were exposed to a biotinylated CRE oligonucleotide. The reaction was run on a 5% native polyacrylamide gel, transferred to a nylon membrane, and the biotinylated oligonucleotide detected by using a streptavidin-horseradish peroxidase antibody and chemiluminescent substrate. Arrow indicates the position of the CREB complex. Lane marked Free is biotinylated oligonucleotide without extract. The bar graph shows relative changes in band intensity for PAECs exposed to estrogen for 16 h. Band intensities were calculated by densitometeric analysis. Binding is expressed as fold changes relative to untreated control. The values expressed are means ± sem, n = 6. *, P < 0.05 vs. untreated; †, P < 0.05 vs. estradiol alone. C, To check the specificity of CREB binding, a competition assay was performed with 50 and 100 times excess of unlabeled CREB oilgonucleotide. There was a decrease in the binding of CREB with 50 and 100 times excess of CREB oligonucleotide. A representative image is shown from four independent experiments. D, Supershift assays were also conducted by incubating nuclear extracts with 4 μg of polyclonal antibodies specific to CREB (CREB Ab) and phosphorylated-CREB (pCREB Ab; 2 h at room temperature). The supershifted complex is shown with an arrow. Panel is a representative image from three independent experiments.

Discussion

Estradiol plays a pivotal role in sexual development and reproduction and is also involved in a number of physiological processes in various tissues including the cardiovascular system. In the present study, we examined the effect of estradiol on GCH1 gene expression in PAECs and elucidated the regulatory mechanisms involved. Our results show that E2 exposure increases GCH1 protein levels, mRNA levels, and promoter activity. We also found that E2 activates the transcription factor CREB and elevates the binding of CREB to the CRE within the GCH1 promoter. These responses can be blocked by the ER inhibitor, ICI 182,780 or eNOS inhibitor, ETU. Together these data indicate that E2-induced GCH1 expression in PAECs involves ER- and eNOS-mediated events. Our data indicate that the signal activating GCH1 expression is membrane initiated because the cell-impermeable estrogen mimic E2-BSA increases GCH1 expression. Furthermore, we demonstrate that the cAMP-PKA signaling pathway is involved in estradiol and NO-induced GCH1 expression. Both ER-α and ER-β appear to be able to transduce the estradiol signal to stimulate GCH1 expression as the transfection of siRNAs specific for both receptors was required to attenuate the increase in GCH1. Finally, our data indicate that the other two genes involved in the de novo pathway of BH4 biosynthesis, PTS and SR, are also increased by estradiol. Together these findings suggest that the estradiol-cAMP-PKA-CREB signaling pathway may stimulate the complete de novo BH4 generation pathway to normalize eNOS activity and allowing it to exert a protective influence on the vascular system (Fig. 6).

Figure 6.

Figure 6

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A potential model for estradiol-induced GCH1 transcription in PAECs. E2 at the cell surface interacts with membrane receptors (ER-α/ER-β) leading to the activation of eNOS and increased NO generation. The NO, either directly or indirectly, leads to the activation of the membrane-associated adenylate cyclase (AC). Activated AC converts ATP to cAMP leading to the dissociation of the inactive tetrameric PKA complex into the active catalytic (C) and the regulatory (R) subunits. The catalytic subunit then shuttles into the nucleus and phosphorylates (P) and activates the transcription factor, CREB. Phosphorylated CREB can then interact with the CRE of the GCH1 gene to activate transcription. Estradiol may also stimulate PTS or SR gene transcription through the same PKA-CREB pathway.

NO is an important mediator of normal vascular tone. Reduced NO synthesis or increased NO consumption by reactive oxygen species results in endothelial dysfunction. NO bioavailability can be increased through augmentation of vascular BH4 levels through pharmacological supplementation (27), reducing BH4 oxidation (28), or enhancing de novo biosynthesis (8). Thus, BH4 levels are a target for therapeutic intervention in vascular diseases (reviewed in Ref. 29). GCH1 is the first and rate-limiting enzyme in the de novo pathway of BH4 biosynthesis (8,9), and studies have demonstrated that overexpression of GCH1 is sufficient to augment BH4 levels in cultured endothelial cells (30). Furthermore, mutations in the GCH1 gene cause BH4 deficiency and are responsible for severe diseases including atypical phenylketonuria and the autosomal dominant 3,4-dihydroxyphenylalanine-responsive dystonia (31). Thus, improved understanding of the mechanisms regulating GCH1 could elicit novel methods of therapeutic intervention.

Studies demonstrate that GCH1 regulation occurs at the transcriptional, posttranscriptional, and posttranslational levels. Several factors, including cytokines, nerve growth factors, cAMP analogs, glucocorticoids, nicotine, and estradiol are reported to influence GCH1 gene expression (32,33,34,35,36). Among these, an increasing number of studies demonstrate that estradiol has favorable effects on vascular cells. Many of these effects are achieved through rapid estrogen receptor-dependent activation of eNOS. The rapid activation of eNOS by estradiol is a key component of both basal and stimulated NO release, significantly affecting vascular homeostasis (reviewed in Ref. 16). The initial observation of estradiol effects on GCH1 gene expression was reported that administration of E2 in vivo increased GCH1 mRNA levels in several catecholaminergic locations (19). Subsequent studies demonstrated that estradiol elevated GCH1 promoter-driven luciferase reporter activity and GCH1 mRNA in cultured PC12 cells, indicating that ERs are involved in estradiol-stimulated GCH1 promoter activity (20,37).

Our data demonstrate that estradiol increases GCH1 protein expression in a time- and dose-dependent manner within endothelial cells. We also found that the estrogen receptor antagonist ICI 182,780 abolishes estradiol-stimulated GCH1 protein expression, consistent with other published results (20,21), and suggests that estradiol-induced GCH1 expression is mediated through ER activation. Furthermore, our data indicate that the activation signal is initiated on the membrane as the membrane-impermeable estradiol form, E2-BSA was able to stimulate GCH1 promoter activity. In addition, both ER-α and ER-β are capable of transducing the signal because an siRNA-mediated decrease in the expression of both receptors was required to attenuate the estradiol-mediated increase in GCH1 promoter activity.

Because estradiol is able to enhance vascular NO bioavailability by increasing eNOS expression and activity (38), we hypothesized that eNOS mediates estradiol- induced GCH1 expression. To test this hypothesis, we examined the effect of eNOS inhibitor ETU on estradiol-stimulated GCH1 expression. Our results show that ETU blocked estradiol-stimulated GCH1 protein expression, thus indicating that eNOS is directly involved in estradiol-stimulated GCH1 expression. Our results are supported by other functional studies: Lam et al. (15) demonstrated that increased blood flow causes coordinated up-regulation of eNOS and GCH1 and that elevation of GCH1 protein expression is accompanied by increased amounts of BH4. Miyazaki-Akita et al. (21) used siRNA ablate eNOS mRNA in bovine aortic endothelial cells and measured the expression levels of GCH1mRNA. They found that the expression of GCH1 is greatly affected by the presence of eNOS. However, they found that even when eNOS expression was attenuated, estradiol was still capable of increasing GCH1 expression levels (21). This suggests that there may also be non-NO-mediated pathways by which estradiol can increase GCH1. Or alternatively, one could argue that there was still enough residual eNOS that NO signaling could still maintain GCH1 expression. Further investigations will be needed to elucidate these possible non-NO mediated regulatory mechanisms.

The second messenger cAMP regulates important cellular functions, including proliferation, differentiation, and apoptosis (39). GCH1 gene expression is regulated by numerous signal transduction pathways, including those that use the second messenger cAMP (32,40). PKA is a key cellular target of second messenger cAMP and CREB is a major nuclear target for the catalytic subunit of PKA. To determine whether PKA was involved in estradiol-stimulated GCH1 expression, we used the PKA inhibitor, H-89, and found that it blocked estradiol-induced increases in GCH1 promoter activity. Forskolin is a rapid and reversible activator of adenylate cyclase, which causes increase in cAMP production (41,42,43). We observed an increase in GCH1 promoter activity with forskolin treatment and an attenuated response when we mutated the CREB binding site at −89, suggesting that cAMP activates GCH1 expression by increasing binding of CREB to CRE-box located in the GCH1 promoter.

CREB is a well-characterized stimulus-induced transcription factor, and the phosphorylation of CREB at the Ser133 site is an important step in the activation of genes with the CRE sites in their promoters (44). We found that the level of phospho-CREB was increased more than 2-fold after 15 min exposure to estradiol and declined to near basal levels by 3 h of treatment. There was a second sustained about 2-fold elevation of phospho-CREB at the 6-h time point for up to 16 h of estradiol exposure. This result is consistent with the time course of GCH1 protein expression induced by estradiol. Our findings regarding the biphasic response of phospho-CREB in response to E2 are supported by previous studies. For example, stimulation of CREB phosphorylation by nicotine or retinoic acid in PC12 cells were sustained with phospho-CREB levels and remained elevated for at least 5 h (22,45), whereas the induction of CRE-mediated gene expression is correlated with sustained, rather than transient phospho-CREB in the nucleus (46). We also examined effects of the ER antagonist, ICI 182,780 and the eNOS inhibitor ETU on estradiol-stimulated sustained phospho-CREB. Our results indicate that both ER and NO signaling are involved in this estradiol-evoked sustained phosphorylation of CREB in PAECs. Other studies have also found that ER activation mediates phosphorylation of CREB (47). However, some reports have demonstrated that estradiol through the direct activation of calcium-dependent kinases, and not ER activation, stimulates phosphorylation of CREB (48). Other studies also found that NO signaling is involved in regulation of phospho-CREB levels. For example, NO has been shown to induce c-fos expression via the phosphorylation of CREB in rat retinal pigment epithelium cells (49), whereas NO was found to elevate phospho-CREB levels in central spinal cord of rats (50), and NOS inhibition has been demonstrated to attenuate the hypoxia-induced increase in phospho-CREB levels in the cerebral cortex of newborn piglets (51).

The human GCH1 core promoter is reported to lie within the first 211 bp upstream from the transcription start site (52). The GCH1 promoter-reporter construct used in our experiments is identical with that used previously (24). In addition, the proximal promoter of the rat and human GCH1 gene contain a sequence motif resembling a CRE, putative CREB binding domain (24,36). Studies have also shown that constitutively active mutant of CREB strongly stimulated GCH1 promoter activity (53), indicating that the CRE in the GCH1 gene is, in fact, functional. Here we have shown that estradiol increases GCH1 promoter activity when an intact CRE motif is present. Mutations within this sequence significantly impaired transcriptional up-regulation by estradiol. Moreover, ER activation and NO signaling may be involved in transcriptional regulation of GCH1 gene by estradiol in PAECs. These data are in agreement with other studies showing transcriptional activation of GCH1 in response to estradiol in PC12 cells or bovine aortic endothelial cells (18,19). Furthermore, our EMSA data indicate that the transcription factor CREB is the likely candidate recruited by the CRE site in the GCH1 promoter because CREB binding increases in response to estradiol treatment. Our data are in agreement with a previous study in human HepG2 hepatoma cells in which it was demonstrated that CREB was required to activate transcription of a GCH1 promoter reporter gene (53).

In summary, our data suggest that in PAECs, ER activation and eNOS-dependent NO signaling are required for estradiol to stimulate GCH1 expression. This involves the phosphorylation and activation of CREB. In addition, the expression of the other two proteins involved in the de novo BH4 biosynthesis, PTS and SR, also appear to be increased by estradiol. Thus, our study provides a novel insight regarding the molecular basis of the regulation of BH4 biosynthesis by estradiol in the pulmonary system.

Acknowledgments

We thank Dr. Gregory Kapatos (Wayne State University, School of Medicine, Detroit, MI) for the kind gift of the GTP cyclohydrolase promoter construct. In addition, the authors thank Dr. Dean Wiseman for critical reading of this manuscript.

Footnotes

This work was supported in part by National Institutes of Health Grants HL60190, HL67841, HL72123, HL084739, and HL70061 (to S.M.B.) and a grant from the Fondation Leducq (to S.M.B.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online April 23, 2009

Abbreviations: BH4, Tetrahydrobiopterin; CRE, cAMP response element; CREB, cAMP response element-binding protein; E2, 17β-estradiol; eNOS, endothelial NOS; ER, estrogen receptor; ETU, 2-ethyl-2-thiopseudourea; GCH1, GTP cyclohydrolase 1; NO, nitric oxide; NOS, NO synthase; NOX, nitrate plus nitrite; PAEC, pulmonary arterial endothelial cell; PKA, protein kinase A; PTS, 6-pyruvoyl tetrahydrobiopterinsynthase; siRNA, small interfering RNA; SR, sepiapterin reductase.

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