Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131-5218
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ABSTRACT |
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The ovarian hormone
17-estradiol (E2
) attenuates chronic hypoxia-induced
pulmonary hypertension. We hypothesized that E2
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 E2
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, E2
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 E2
-induced
attenuation of pulmonary ET-1 expression during hypoxia results from
decreased HIF activity. We found that E2
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 E2
-mediated attenuation of
hypoxia-induced ET-1 promoter activity. We conclude that
E2
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 17-estradiol (E2
) during CH. However, the mechanisms by which E2
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
E2 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
E2
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 E2
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
E2
-mediated attenuation of hypoxia-induced ET-1
expression is the result of competition for CBP/p300.
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METHODS |
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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 E2 (0.8 µg/h via subcutaneous osmotic pumps); and 4) CH OVX rats
that received the vehicle for E2
(97% 1,2 propanediol-3% ethanol). Additional normoxic OVX vehicle- and
E2
-treated groups were included for assessments of lung
ET-1 peptide levels. Previous findings from our laboratory have
demonstrated that this dose of E2
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 E2
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 E2 (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 [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 E2 (10 nM) or its vehicle (ethanol) to determine the
effect of E2
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 E2 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 E2
(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 E2
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 E2 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 E2
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. ![]() |
RESULTS |
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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 E2
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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 E2 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, E2
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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
E2 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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Reporter Gene Experiments
ET-1 promoter activity.
Administration of E2 (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 E2
(Fig. 3A). ET-1
promoter activity was not altered by either hypoxia or
E2
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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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, E2 (1 or 10 nM) abolished hypoxia-induced HRE reporter activity (Fig. 4A).
However, E2
administration (10 nM) to normoxic cells
transfected with the HRE construct also decreased luciferase activity
(Fig. 4A). Neither hypoxia nor E2
(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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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, E2 (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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DISCUSSION |
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The effects of the ovarian hormone E2 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) E2
replacement to OVX rats prevents increases in pulmonary preproET-1 mRNA
and ET-1 peptide levels induced by 24-h hypoxic exposure; 3)
E2
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 E2
on hypoxia-induced ET-1
promoter activity. Together, these findings suggest that
E2
attenuates hypoxic induction of ET-1 gene expression
by interfering with HIF activity and that these inhibitory effects of
E2
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 E2
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
E2
replacement. These findings suggest that
E2
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
E2. The reason for these apparent differences is not clear but could reflect normal fluctuations in plasma E2
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 E2
replacement prevents hypoxic induction of pulmonary ET-1 gene and peptide expression, results from cultured cell studies suggest that
these responses to E2
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, E2
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
E2
administration. Because hypoxia-induced increases in
reporter activity of this construct are strictly HIF dependent,
opposition of HIF activity by E2
appears to account for
diminished hypoxia-induced ET-1 gene expression. However, these
experiments do not rule out the possibility that E2
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 E2 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 E2
-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 E2
replacement in CH OVX rats
(26). Therefore, it is unlikely that increased production of NO accounts for the inhibitory effects of E2
on
hypoxia-induced ET-1 expression within the lung.
Further experiments were performed to determine at what point
E2 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, E2
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 E2
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 E2
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 E2
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,
E2 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 E2
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 E2
(1 nM). These data suggest
that decreased hypoxia-induced EPO gene expression after
E2
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 E2
(24).
In summary, we have demonstrated that hypoxia-induced increases in
pulmonary ET-1 gene expression are attenuated by the ovarian hormone
E2. Furthermore, our findings suggest that
E2
attenuates HIF-1-mediated increases in both ET-1 and
EPO gene expression. The inhibitory effects of E2
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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