Estradiol attenuates hypoxia-induced pulmonary endothelin-1 gene expression

Scott Earley and Thomas C. Resta

Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131-5218


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The ovarian hormone 17beta -estradiol (E2beta ) attenuates chronic hypoxia-induced pulmonary hypertension. We hypothesized that E2beta attenuates this response to hypoxia by decreasing pulmonary expression of the vasoactive and mitogenic peptide endothelin-1 (ET-1). To test this hypothesis, we measured preproET-1 mRNA and ET-1 peptide levels in the lungs of adult female normoxic and hypoxic (24 h or 4 wk at barometric pressure = 380 mmHg) rats with intact ovaries and in hypoxic ovariectomized (OVX) rats administered E2beta or vehicle via subcutaneous osmotic pumps. Hypoxic exposure increased lung preproET-1 mRNA levels in OVX vehicle-treated rats, but not in rats with intact ovaries. In addition, E2beta replacement prevented hypoxia-mediated increases in preproET-1 mRNA and ET-1 peptide expression. Considering that hypoxic induction of ET-1 gene expression is mediated by a hypoxia-inducible transcription factor(s) (HIF), we further hypothesized that E2beta -induced attenuation of pulmonary ET-1 expression during hypoxia results from decreased HIF activity. We found that E2beta abolished HIF-dependent increases in reporter gene activity. Further experiments demonstrated that overexpression of the transcriptional coactivator cAMP response element binding protein (CREB) binding protein (CBP)/p300, a factor common to both the estrogen receptor and HIF pathways, eliminated E2beta -mediated attenuation of hypoxia-induced ET-1 promoter activity. We conclude that E2beta inhibits hypoxic induction of ET-1 gene expression by interfering with HIF activity, possibly through competition for limiting quantities of CBP/p300.

pulmonary hypertension; hypoxia-inducible factor; reporter gene activity


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INTRODUCTION
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CHRONIC HYPOXIA (CH) is a consequence of prolonged residence at high altitude and pathological conditions that impair oxygenation of the blood, such as chronic obstructive pulmonary disease (COPD). Physiological responses to CH include hypoxic pulmonary vasoconstriction (HPV), pulmonary arterial remodeling, and polycythemia. Increased pulmonary vascular resistance and the resultant pulmonary hypertension associated with these responses lead to right ventricular hypertrophy and right heart failure. Epidemiological studies suggest that women with COPD exhibit a decreased risk of mortality compared with men (30), indicating that gender-specific factors may influence the development of hypoxia-induced pulmonary hypertension. In agreement with these observations, a previous study from our laboratory (26) has demonstrated that CH ovariectomized (OVX) rats develop more severe right ventricular hypertrophy and pulmonary arterial remodeling than either CH rats with intact ovaries or CH OVX rats administered the ovarian hormone 17beta -estradiol (E2beta ) during CH. However, the mechanisms by which E2beta exerts such protective influences in the hypertensive pulmonary circulation have yet to be clarified.

The endothelium-derived vasoactive and mitogenic peptide endothelin-1 (ET-1) appears to play a critical role in the development of CH-induced pulmonary hypertension. For example, endothelin A (ETA) receptor blockade attenuates hypoxia-induced pulmonary arterial remodeling in male rats (5), and the vasoconstrictor properties of ET-1 may augment HPV (27). Furthermore, pulmonary ET-1 synthesis and gene expression are elevated with hypoxic exposure (6). We therefore hypothesized that E2beta attenuates hypoxia-induced pulmonary hypertension by decreasing ET-1 gene expression within the lung. To test this hypothesis, we measured preproET-1 mRNA and ET-1 peptide levels in lungs from CH rats with intact ovaries and OVX rats administered E2beta or its vehicle. Considering that hypoxic induction of ET-1 gene expression appears to require the transcription factor hypoxia-inducible factor 1 (HIF-1) (10, 31), we further hypothesized that attenuation of hypoxia-induced pulmonary ET-1 expression by E2beta results from decreased HIF-1 activity. Interestingly, the transcriptional coactivator cAMP response element binding protein (CREB) binding protein (CBP)/p300 is required for transactivation by both HIF (1) and estrogen receptor (9, 19) signaling pathways. Considering that CBP/p300 has been shown to act as a cointegrator of multiple transcriptional pathways (15), we also tested the hypothesis that E2beta -mediated attenuation of hypoxia-induced ET-1 expression is the result of competition for CBP/p300.


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Intact Animal Experiments

All animal protocols and surgical procedures employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine (Albuquerque, NM).

Experimental animal groups. To assess the effects of estrogen on hypoxia-induced pulmonary ET-1 gene and peptide expression, four groups of female Sprague-Dawley rats (200-350 g; Harlan Industries) were prepared: 1) normoxic rats with intact ovaries; 2) CH rats with intact ovaries; 3) CH OVX rats that received E2beta (0.8 µg/h via subcutaneous osmotic pumps); and 4) CH OVX rats that received the vehicle for E2beta (97% 1,2 propanediol-3% ethanol). Additional normoxic OVX vehicle- and E2beta -treated groups were included for assessments of lung ET-1 peptide levels. Previous findings from our laboratory have demonstrated that this dose of E2beta provides plasma levels of the hormone within the physiological range and attenuates CH-induced right ventricular hypertrophy and pulmonary arterial remodeling in OVX rats (26). Animals designated for exposure to CH were housed in a hypobaric chamber with barometric pressure maintained at ~380 Torr. To discriminate between the effects of short- and long-term chronic hypoxia, two hypoxic exposure protocols were employed for this study: one group of rats was exposed to hypoxia for 24 h, whereas hypoxia was maintained for the second group for 4 wk. The 24-h hypoxic exposure protocol was used to match the duration of hypoxic exposure employed for cultured cell studies. In addition, evidence of arterial remodeling has been detected after 24 h of hypoxic exposure in rat lungs (28), suggesting that the shorter time point is relevant to the onset of pulmonary vascular remodeling and the development of pulmonary hypertension. The hypobaric chamber was opened three times per week during the 4-wk exposure period to provide animals with fresh food, water, and clean bedding. On the day of experimentation, animals were removed from the hypobaric chamber and immediately placed in Plexiglas chambers continuously flushed with a 12% O2-88% N2 gas mixture to reproduce inspired PO2 (~70 mmHg) within the hypobaric chamber. Age-matched normoxic control animals were housed at ambient barometric pressure (~630 mmHg). Separate normoxic control groups were maintained for each hypoxic exposure protocol. All animals were maintained on a 12:12-h light-dark cycle. At the end of the hypoxic exposure period, the animals were anesthetized with pentobarbital sodium (32.5 mg ip), and the lungs were harvested and snap-frozen in liquid N2. In addition, blood samples were collected by direct cardiac puncture for measurement of hematocrit, and uterine weight was assessed as an index of E2beta delivery (26).

Surgical procedures for ovariectomy and osmotic pump implantation. Rats designated for OVX were anesthetized with a mixture of ketamine (90 mg/kg im) and acepromazine (0.9 mg/kg im). With sterile technique, ovaries were resected through bilateral flank incisions. Rats were allowed at least 2 wk to recover before implantation of osmotic pumps (Alzet model 2ML4 for 4-wk protocols and model 2002 for 24-h protocols) for administration of E2beta (Sigma) or vehicle. Osmotic pumps were implanted subcutaneously via a midline incision between the scapulae in rats anesthetized with ketamine-acepromazine. All animals were administered systemic and topical antibiotics postoperatively. Rats designated for 4-wk hypoxic exposure were placed in the hypobaric chamber the morning after osmotic pump implantation. Rats designated for 24-h hypoxic exposure were allowed to recover for 1 wk after osmotic pump implantation before being placed in the hypobaric chamber.

Ribonuclease Protection Assay for preproET-1 mRNA

Total RNA was prepared from snap-frozen rat lungs using TRIzol (Life Technologies) reagent. cDNA was reverse transcribed from total rat lung RNA in reactions containing 0.1 µg/µl of RNA, 10 µM oligo(dT)16 (Perkin-Elmer), 200 µM each dNTP, and 20 units of avian myeloblastosis virus reverse transcriptase (Promega). A ribonuclease protection assay (RPA) probe template for ET-1 was constructed using PCR primers 5'-GAACTCCGAGCCCAAAGTAC-3' (forward) and 5'-CTTGCTAAGATCCCAGCCA-3' (reverse) based on a published rat ET-1 mRNA sequence (GenBank accession no. M64711). Typical PCR condition reaction consisted of 5 µl of rat cDNA, 5 units of Pyrococcus furiosus DNA polymerase (Stratagene), 0.1-0.5 µM each primer, and 100-250 µM each dNTP. The 321-bp PCR product was confirmed by sequencing. PCR products were used to generate probe templates by reamplifying the products in PCR reactions using a reverse primer that had the sequence for the T7 RNA polymerase promoter (5'-TAATACGACTCACTATAGGGAGGA-3') added to the 5' end of the original reverse primer. This reaction incorporated the T7 promoter sequence into the new PCR products in an orientation that allowed for the expression of the antisense strand. Radiolabeled antisense RNA (cRNA) was prepared by incubating 0.5 µg of template DNA in the presence of 10 units of T7 RNA polymerase and 50 µCi of [alpha -32P]UTP (800 Ci/mmol, 20 mCi/ml, Amersham) using the MAXIscript in vitro transcription kit (Ambion). RPA was performed using reagents supplied by the RPA III kit (Ambion). Aliquots of the labeled probes containing 5 × 104 counts/min of ET-1 cRNA were mixed with 10 µg of total RNA and a molar excess of a trace-labeled 18S rRNA probe (Ambion, pTRI RNA 18S) and incubated overnight at 42°C. Hybridization reactions were digested with a mixture of RNase T1 and RNase A and electrophoresed through denaturing polyacrylamide gels. The dried gels were used to expose Phosphor Storage Screens (Molecular Dynamics), the screens were scanned with a STORM 860 PhosphorImager (Molecular Dynamics), and the appropriate bands were quantitated using ImageQuant software. 18S rRNA was used as a constitutively expressed internal control for RNA quantity and quality. ET-1 mRNA abundance was determined by dividing the band volumes for ET-1 by those of the corresponding 18S rRNA bands. Additional hybridizations containing 2.5, 5, or 10 µg of RNA from hypoxic rat lung were performed and subjected to RPA to demonstrate the linearity of the relationship between ET-1 and 18S rRNA band volumes and input RNA quantity.

Measurement of ET Peptide in Lung Tissue

Snap-frozen lung tissue was homogenized using a Polytron blender in ice-cold methanol. After a brief centrifugation, the extract was purified and concentrated using reverse-phase Amprep C2 columns. Lung ET peptide levels were determined using an RIA kit (Peninsula Laboratories) and were expressed as picograms of ET per milligram of extracted lung tissue. The antibody provided with this kit cross-reacts with endothelin-2 (7%) and endothelin-3 (7%).

General Methods: Reporter Gene Experiments

Cell culture. First-passage bovine pulmonary artery endothelial cells (BPAECs; Clonetics) were cultured at 37°C, 6% CO2, balance air in humidified incubators in phenol red-free Endothelial Growth Medium (EGM; Clonetics) supplemented with 2% charcoal-dextran-filtered fetal bovine serum (Hyclone). Cells were passaged with 0.025% trypsin-EDTA when confluent. Second-passage cells were used for all reporter gene experiments. Hypoxic exposures were performed in a Napco 7000 series three-gas incubator at 37°C, 6% CO2, 1% O2, balance N2. Chamber oxygen concentration was verified using an Ametek model S-3A/I oxygen analyzer.

Plasmids. A segment of the preproET-1 promoter containing a functional hypoxia response element (HRE) and other response elements was cloned from rat genomic DNA into a luciferase reporter vector. A 745-bp promoter fragment was amplified by PCR using primers 5'-TAGGATGTGCCTGACGAAAC-3' (forward) and 5'-AGACCCAGTCAGGCTCTCAG-3' (reverse) that were identified from a published sequence (GenBank accession no. S76970). The amplified fragment was cloned into the SrfI site of pPCR-Script-Amp+ (Stratagene), and the orientation of the insert was determined by restriction mapping. An SstI-HindIII fragment containing the ET-1 promoter was cloned into the corresponding sites of the firefly luciferase reporter vector pGL2-basic (Promega), and the resulting plasmid was designated pGL2-ET1P. The identity and orientation of the insert was confirmed by sequencing. pRL-TK (Promega), which expresses Renilla reniformis luciferase under the control of the minimal herpes simplex virus thymidine kinase promoter, was used as an internal control for cell viability and transfection efficiency. Plasmids pEpoE-luc and pEpoEm1-luc were kindly provided by Drs. H. Franklin Bunn and L. Eric Huang. pEpoE-luc (11) consists of a luciferase reporter gene driven by a cloned fragment of the human erythropoietin (EPO) 3' enhancer region containing a functional HRE and the SV40 promoter. The HRE of pEpoE-luc was mutated to a sequence that does not bind HIF to create pEpoEm1-luc (11). pRc/RSV-CBP, which encodes the transcriptional coactivator CBP/p300 (7), was the gift of Dr. Richard Goodman.

Site-directed mutagenesis. Site-directed mutation of the HRE (5'-ACGTGC-3') within the cloned ET-1 promoter fragment was performed using a Quick-change site-directed mutagenesis kit (Stratagene). The sequence of the mutagenic primer was 5'-GGGTCTTATCTCCGGCTGCATACTGCCTGTGGGTGACTAATC-3'. Incorporation of this sequence by P. furiosus PCR into pGL2-ET1P followed by DpnI digestion (to remove template DNA) altered the sequence of the ET-1 promoter HRE to 5'-ATACGC-3'. HIF-1 does not bind this sequence (10) in gel shift assays. The mutation was confirmed by sequencing.

Transfections and reporter gene assays. Transfections of BPAECs were performed using Superfect transfection reagent (Qiagen). BPAECs were split into six-well plates at a density of 100,000 cells/well. On the following day, cells were washed and exposed to a total of 1 µg of reporter plasmid DNA and 5 µl of Superfect reagent in 600 µl of EGM. pRL-TK DNA was cotransfected with reporter plasmid DNA at a ratio of 1:5. Cells were washed, and fresh EGM was replaced after a 4-h incubation period. Transfected cells were incubated for an additional 24 h before experimental treatments. Cells were exposed to experimental treatments for 24 h before passive lysis and luciferase assay. Reporter plasmid activity was determined by the dual-luciferase assay (Promega). Luciferase measurements were performed using a Turner Designs model 20 luminometer. Relative promoter activity in cell lysates was determined by dividing the luminescence observed after the addition of firefly luciferase substrate by that obtained after quenching firefly luciferase activity and adding the substrate for Renilla luciferase. The mean background luminescence from six mock-transfected samples was determined for each experiment and was subtracted from each sample before ratio calculation.

Experimental Protocols: Reporter Gene Experiments

ET-1 promoter activity. The cloned ET-1 promoter fragment employed for these studies contains endothelial cell-specific response elements (3), resulting in very low activity in nonendothelial cells. Therefore, BPAECs were used for reporter gene experiments. BPAECs transfected with the ET-1 promoter reporter gene construct were cultured under normoxic or hypoxic (1% O2, 24 h) conditions in the presence of E2beta (10 nM) or its vehicle (ethanol) to determine the effect of E2beta on hypoxia-induced increases in promoter activity. Parallel experiments were performed in cells transfected with an ET-1 promoter gene construct that had the HRE within the cloned ET-1 promoter fragment mutated to a sequence that does not bind HIF-1 (10).

HRE-mediated reporter gene activity. The cloned ET-1 promoter fragment employed for reporter gene experiments contains multiple putative response elements (3, 31) in addition to the HIF-binding site. Therefore, additional reporter gene experiments were performed to determine whether E2beta inhibits HIF activity per se or, rather, interferes with the activity of other transcription factors that may be required for HIF-mediated transcription. The luciferase reporter gene of pEpoE-luc is driven by an HRE from the human EPO 3'-enhancer element and an SV40 promoter. Thus hypoxia-induced increases in reporter activity of this plasmid are strictly HIF dependent. BPAECs transfected with pEpoE-luc were cultured under normoxic or hypoxic conditions, and the effects of E2beta (1 or 10 nM) or ethanol vehicle on HRE-dependent promoter activity were evaluated. Other cultures were transfected with the plasmid pEpoEm1-luc, identical to pEpoE-luc except for a mutation within the HRE that prevents HIF binding. Effects of hypoxia (1% O2, 24 h) and E2beta on pEpoEm1-luc reporter activity were similarly evaluated.

CBP/p300 overexpression. The estrogen receptor and HIF pathways both require the transcriptional coactivator CBP/p300 for full transcriptional activity. To determine whether the inhibitory effects of E2beta are due to competition between ligand-activated estrogen receptor and HIF for limiting quantities of this factor, we transfected cells with 1 µg of the ET-1 promoter vector as well as 1 µg of pRc/RSV-CBP, which contains the gene for CBP/p300 under the control of the Rous sarcoma virus promoter. BPAECs transfected with both plasmids were cultured under normoxic or hypoxic (1% O2, 24 h) conditions, and the effects of E2beta administration (10 nM) on ET-1 promoter activity was evaluated.

Calculations and Statistics

All data are expressed as means ± SE. Values of n refer to the number of animals or the number of replicate cultures in each group. One-way analysis of variance (ANOVA) was used to make comparisons. If differences were detected by ANOVA, individual groups were compared using the Student-Newman-Keuls test. Data expressed as percentages were normalized using the arcsine transformation before statistical analysis. A level of P < 0.05 was accepted as statistically significant for all comparisons.


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Uterine Weights and Hematocrit

Uterine weights were not different between normoxic and 24-h hypoxic rats with intact ovaries but were significantly decreased for hypoxic OVX vehicle-treated rats compared with intact groups as expected (Table 1). Uterine weights of hypoxic OVX rats receiving E2beta replacement were greater than both hypoxic OVX vehicle-treated rats and normoxic rats with intact ovaries but were not different from intact hypoxic rats (Table 1). Similarly, uterine weights were not different between normoxic and 4-wk CH rats with intact ovaries but were significantly decreased for CH OVX vehicle-treated rats compared with intact groups (Table 1). Uterine weights of CH OVX rats receiving E2beta replacement were greater than CH OVX vehicle-treated rats but were significantly less than those of intact rats (Table 1).

                              
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Table 1.   Uterine weights

Hematocrit was increased after 4 wk of hypoxic exposure (61 ± 1%) compared with normoxic controls for rats with intact ovaries (40 ± 1%). Ovariectomy exacerbated hypoxia-induced polycythemia (68 ± 1%), whereas E2beta replacement to OXV rats attenuated hypoxia-induced increases in hematocrit (54 ± 2%), as we have previously reported (26).

PreproET-1 mRNA and ET Peptide Levels in Rat Lung

PreproET-1 mRNA levels were greater in lungs from OVX vehicle-treated rats exposed to hypoxia for 24 h compared with normoxic and hypoxic rats with intact ovaries (Fig. 1A). Interestingly, E2beta replacement to OVX rats abolished hypoxic induction of preproET-1 mRNA (Fig. 1A). Consistent with these findings, ET peptide levels were also elevated in lungs from OVX vehicle-treated hypoxic rats compared with normoxic intact rats (Fig. 1B). Furthermore, E2beta replacement to hypoxic OVX rats prevented increases in ET peptide levels (Fig. 1B). In contrast, pulmonary ET peptide levels did not differ between normoxic OVX vehicle-treated rats (2.02 ± 0.20 pg/mg lung, n = 6) and normoxic OVX rats that received E2beta replacement (1.95 ± 0.14 pg/mg lung, n = 6).


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Fig. 1.   Twenty-four-hour hypoxia protocol. Prepro-endothelin-1 (ET-1) mRNA (A) and ET peptide (B) levels in lungs from each group; n = 6 for all groups. * P <=  0.05 vs. normoxia intact; # P <=  0.05 vs. hypoxia intact; dagger  P <=  0.05 vs. hypoxia OVX VEH. OVX, ovariectomized; VEH, vehicle; E2beta , 17beta -estradiol.

A somewhat similar pattern of lung ET expression was observed after 4 wk of hypoxic exposure in OVX rats. Specifically, whereas hypoxia increased pulmonary ET peptide levels in CH OVX vehicle-treated rats compared with normoxic intact rats (Fig. 2, A and B), no such effect of hypoxia was observed for animals receiving E2beta replacement. Although CH tended to increase preproET-1 mRNA and ET peptide levels in intact animals, significance was achieved only for ET peptide.


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Fig. 2.   Four-week hypoxia protocol. PreproET-1 mRNA (A) and ET peptide (B) levels in lungs from each group; n = 6 for all groups. * P <=  0.05 vs. normoxia intact.

Reporter Gene Experiments

ET-1 promoter activity. Administration of E2beta (10 nM) to transfected BPAECs under normoxic conditions had no effect on ET-1 promoter activity (Fig. 3A). In contrast, when transfected cells were cultured under hypoxic conditions, ET-1 promoter activity was increased compared with normoxic controls, and this increase was abolished by E2beta (Fig. 3A). ET-1 promoter activity was not altered by either hypoxia or E2beta administration for cells transfected with a reporter plasmid in which the ET-1 promoter HRE had been mutated to a sequence that does not bind HIF (Fig. 3B).


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Fig. 3.   A: ET-1 promoter activity in bovine pulmonary arterial endothelial cells (BPAECs) cultured under hypoxic (1% O2, 24 h) or normoxic (21% O2) conditions in the presence of E2beta (10 nM) or vehicle. N = 6 for normoxia vehicle, normoxia E2beta , and hypoxia E2beta . N = 3 for hypoxia vehicle. * P <=  0.05 vs. all other groups. B: hypoxia response element (HRE)-mutant ET-1 promoter activity in BPAECs cultured under hypoxic or normoxic conditions in the presence of E2beta (10 nM) or vehicle; n = 6 for all groups. There are no significant differences.

HRE-mediated reporter activity. HRE-mediated reporter gene activity was greater in BPAECs exposed to hypoxia compared with normoxic controls (Fig. 4A). Similar to results from ET-1 reporter gene experiments, E2beta (1 or 10 nM) abolished hypoxia-induced HRE reporter activity (Fig. 4A). However, E2beta administration (10 nM) to normoxic cells transfected with the HRE construct also decreased luciferase activity (Fig. 4A). Neither hypoxia nor E2beta (10 nM) altered the reporter activity of cells transfected with a similar construct containing an HRE altered by site-specific mutagenesis to a sequence that does not bind HIF (Fig. 4B).


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Fig. 4.   A: HRE-mediated reporter activity in BPAECs cultured under hypoxic (1% O2, 24 h) or normoxic (21% O2) conditions in the presence of E2beta (1 or 10 nM) or vehicle; n = 6 for all groups. * P <=  0.05 vs. normoxia vehicle. # P <=  0.05 vs. hypoxia vehicle. B: HRE-mutant reporter activity in BPAECs cultured under hypoxic or normoxic conditions in the presence of E2beta (10 nM) or vehicle; n = 6 for all groups. There are no significant differences.

CBP/p300 overexpression. ET-1 promoter activity was increased by hypoxic exposure for cells transfected with both the ET-1 reporter vector and a plasmid (pRc/RSV-CBP) expressing the transcriptional coactivator CBP/p300 (Fig. 5). However, E2beta (10 nM) had no effect on ET-1 promoter activity in cells transfected with pRc/RSV-CBP under either hypoxic or normoxic conditions (Fig. 5).


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Fig. 5.   ET-1 promoter activity in BPAECs cotransfected with a plasmid (pRc/RSV) that expresses cAMP response element binding protein binding protein (CBP)/p300. Transfected cells were cultured under hypoxic (1% O2, 24 h) or normoxic (21% O2) conditions in the presence of E2beta (10 nM) or vehicle; n = 6 for all groups. * P <=  0.05 vs. normoxia vehicle and normoxia E2beta .


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of the ovarian hormone E2beta on hypoxia-induced ET-1 gene expression were examined in rat lung as well as a reporter gene system employing cultured pulmonary artery endothelial cells. The major findings of this study are: 1) hypoxia increases preproET-1 mRNA levels in lungs of OVX rats but not in lungs of rats with intact ovaries; 2) E2beta replacement to OVX rats prevents increases in pulmonary preproET-1 mRNA and ET-1 peptide levels induced by 24-h hypoxic exposure; 3) E2beta eliminates hypoxia-dependent increases in ET-1 promoter and HRE-mediated reporter gene activity in BPAECs; and 4) transfection of endothelial cells with a plasmid that expresses the transcriptional coactivator CBP/p300 abolishes the inhibitory effects of E2beta on hypoxia-induced ET-1 promoter activity. Together, these findings suggest that E2beta attenuates hypoxic induction of ET-1 gene expression by interfering with HIF activity and that these inhibitory effects of E2beta may result from competition between the estrogen receptor and HIF pathways for limiting amounts of CBP/p300.

Hypoxia-induced pulmonary hypertension results from increased vascular resistance associated with HPV, polycythemia, and pulmonary vascular remodeling. A previous study from our laboratory demonstrated that OVX exacerbated the development of CH-induced right ventricular hypertrophy and pulmonary arterial remodeling, whereas E2beta replacement prevented the effects of OVX (26). These findings are consistent with the results of clinical studies indicating a lower incidence of pulmonary hypertension among female vs. male COPD patients (30) and other studies demonstrating a sexually dimorphic pattern in the development of hypoxia-induced pulmonary hypertension in chickens (4), swine (22), and rats (25). Chronic administration of ETA antagonists attenuates the severity of hypoxia-induced pulmonary vascular remodeling in male rats (5), suggesting that the mitogenic properties of ET-1 may be critically important for this process. Several studies have demonstrated that preproET-1 mRNA and ET peptide levels in rat lung tissue and cultured endothelial cells are elevated after hypoxic exposure (6, 8, 18, 20), further suggesting that increased pulmonary ET-1 synthesis may contribute to hypoxia-induced pulmonary arterial remodeling, HPV, and associated pulmonary hypertension. Results from the present study indicate that OVX augments hypoxic induction of pulmonary preproET-1 gene expression and ET peptide levels and that this response to OVX is attenuated by E2beta replacement. These findings suggest that E2beta moderates the development of pulmonary hypertension by interfering with increased pulmonary ET-1 gene and peptide expression during hypoxic exposure.

It is noteworthy that, in contrast with hypoxic rats, ET peptide levels tended to be elevated in CH rats with intact ovaries compared with intact normoxic controls as well as CH OVX rats receiving E2beta . The reason for these apparent differences is not clear but could reflect normal fluctuations in plasma E2beta levels that occur during the 4-day estrus cycle in rats with intact ovaries vs. continuous estradiol administration via osmotic pumps. In addition, the differences in pulmonary ET-1 peptide levels between hypoxic and CH rats may be related to physiological adaptation that occurs during prolonged hypoxic exposure. Tissue oxygen delivery may be greater after 4 wk of hypoxia compared with 24 h due to these adaptive responses. Therefore, it is possible that HIF-dependent responses are more important in regulating ET-1 expression during the earlier phases of hypoxic exposure, whereas secondary effects, such as increased shear stress, may be more relevant during prolonged hypoxic exposure. However, evidence of pulmonary arterial remodeling has been observed within 24 h of hypoxic exposure in rats (28), suggesting that remodeling commences soon after the initiation of hypoxia. Therefore, decreased ET-1 levels in the preadaptive stage of hypoxia could delay the onset of pulmonary vascular remodeling. It is also not immediately apparent why ET-1 peptide levels increased in CH intact rats in the absence of a significant increase in message levels, although it is possible that such divergent responses result from posttranslational modifications in ET-1 synthesis or peptide stability. Alternatively, if lung preproET-1 mRNA levels coincide with fluctuations in plasma estrogen during the estrus cycle, it is conceivable that mRNA levels were declining at a point when ET-1 peptide levels remained elevated.

Consistent with our findings from whole animals that E2beta replacement prevents hypoxic induction of pulmonary ET-1 gene and peptide expression, results from cultured cell studies suggest that these responses to E2beta are a function of decreased ET-1 promoter activity. Furthermore, hypoxia-induced increases in ET-1 promoter activity were HRE dependent. Although an early report demonstrated that hypoxia-induced increases in ET-1 gene expression result from the activity of the transcription factor HIF-1 (10), a more recent work shows that the HIF-1 binding site alone is not sufficient for the transcriptional response to hypoxia (31). This latter study demonstrated that binding sites for activator protein-1 (AP-1), GATA-2, and CAAT-binding factor are also required for hypoxic induction of ET-1 promoter activity. Interestingly, E2beta also attenuates serum-induced ET-1 production by cultured endothelial cells under normoxic conditions, possibly by interfering with AP-1 activity (23). Our present findings demonstrated that hypoxia-induced increases in HRE-mediated reporter gene activity were also attenuated by E2beta administration. Because hypoxia-induced increases in reporter activity of this construct are strictly HIF dependent, opposition of HIF activity by E2beta appears to account for diminished hypoxia-induced ET-1 gene expression. However, these experiments do not rule out the possibility that E2beta may interfere with a non-HIF factor that is required for hypoxic induction of either the ET-1 promoter or the EPO enhancer region. Furthermore, the correlation between the in vivo findings and the reporter gene data should be interpreted cautiously because these studies were performed in different species (rat vs. bovine), and signaling pathways may not be identical between the two models.

Several studies have demonstrated that E2beta may increase vascular nitric oxide (NO) production (2, 16, 17, 21). NO donors reportedly attenuate HIF-dependent gene expression in cultured hepatoma cell lines (12, 29), suggesting that E2beta -induced increases in NO production may account for diminished ET-1 expression during hypoxia. However, a recent study from our laboratory demonstrated that inhaled NO had no effect on ET-1 expression in the lung during hypoxic exposure, and NO donors did not attenuate hypoxia-induced ET production in cultured pulmonary artery endothelial cells (6). Furthermore, neither pulmonary endothelial nor inducible nitric oxide synthase expression is increased by E2beta replacement in CH OVX rats (26). Therefore, it is unlikely that increased production of NO accounts for the inhibitory effects of E2beta on hypoxia-induced ET-1 expression within the lung.

Further experiments were performed to determine at what point E2beta might interrupt the HIF pathway and attenuate hypoxia-induced ET-1 gene expression. Control of HIF signaling involves several levels of regulation, including increased protein stability and nuclear translocation under hypoxic conditions (13, 14) and hypoxia-specific recruitment of the transcriptional coactivator CBP/p300 (7). Therefore, E2beta potentially interferes with HIF signaling at several points, for example, by attenuating HIF gene expression or decreasing protein stability. Interestingly, CBP/p300 is also recruited by ligand-activated estrogen receptors to form fully functional transcriptional complexes (9, 19). Kamei et al. (15) have demonstrated that competition for limiting quantities of this factor results in decreased AP-1-dependent transcriptional activity when nuclear receptors are activated by ligand, suggesting that CBP/p300 serves as an integrator of signaling pathways within the nucleus. Therefore, we tested the hypothesis that the inhibitory effects of E2beta on hypoxia-induced gene expression are mediated through competition between the estrogen receptor and HIF pathways for limiting quantities of CBP/p300. Our findings suggest that CBP/p300 overexpression in BPAECs prevents inhibitory influences of E2beta on hypoxic stimulation of ET-1 promoter activity. Although it is possible that other products or activities of the CBP/p300-expressing plasmids could be responsible for the observed effects, these data suggest that inhibition of ET-1 gene expression by E2beta is due to competitive cross talk between the HIF and estrogen receptor pathways for limiting quantities of CBP/p300.

In addition to attenuating pulmonary ET-1 production during hypoxia, E2beta may also inhibit the expression of other HIF-1 responsive genes. For example, in agreement with a previous report from our laboratory (26), our findings demonstrate that OVX augments the development of polycythemia during chronic hypoxic exposure and that this effect of OVX is prevented by E2beta replacement. Elevated levels of EPO resulting from HIF-1-dependent increases in gene expression appear to account for hypoxia-induced polycythemia. Our findings (Fig. 4) demonstrate that reporter gene activity driven by the EPO hypoxia-inducible enhancer element is increased by hypoxic exposure, and this increase is abolished by physiological levels of E2beta (1 nM). These data suggest that decreased hypoxia-induced EPO gene expression after E2beta replacement may account for the observed attenuation of the polycythemic response. Consistently, a preliminary report from our laboratory suggests that hypoxia-induced renal EPO gene expression in OVX rats is reduced in animals receiving E2beta (24).

In summary, we have demonstrated that hypoxia-induced increases in pulmonary ET-1 gene expression are attenuated by the ovarian hormone E2beta . Furthermore, our findings suggest that E2beta attenuates HIF-1-mediated increases in both ET-1 and EPO gene expression. The inhibitory effects of E2beta on hypoxia-induced ET-1 gene expression may be the result of competition between the HIF and estrogen receptor pathways for limiting quantities of the transcriptional coactivator CBP/p300.


    ACKNOWLEDGEMENTS

We thank Anna Holmes and Minerva Murphy for technical assistance, Dr. Ivan F. McMurtry for helpful discussions that contributed to the initial idea for this study, Dr. John Omdahl for aid with site-specific mutagenesis procedures, Drs. H. Franklin Bunn and L. Eric Huang for providing pEpoE-luc and pEpoEm1-luc, Dr. Richard Goodman for supplying pRc/RSV-CBP, and the Center for Genetics in Medicine of the University of New Mexico Department of Biochemistry and Molecular Biology for DNA sequencing services and PCR primer synthesis.


    FOOTNOTES

This work was supported by a Scientist Development Grant from the American Heart Association (T. C. Resta) and by National Institutes of Health National Center for Research Resources Grant RR-164808 (T. C. Resta).

T. C. Resta is a Parker B. Francis Fellow in Pulmonary Research.

Address for reprint requests and other correspondence: S. Earley, Vascular Physiology Group, Dept. of Cell Biology and Physiology, Univ. of New Mexico HSC, 915 Camino de Salud NE, Albuquerque, NM 87131-5218 (E-mail: searley{at}unm.edu).

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

First published February 8, 2002;10.1152/ajplung.00476.2001

Received 13 December 2001; accepted in final form 4 February 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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