Departments of 1 Cardiovascular Biochemistry, 5 Experimental Pathology, and 4 Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543; 2 Vascular Biology and Hypertension Program, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 3 Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Significant
elevations in endothelin (ET)-1 levels accompany many diseases, but the
underlying regulatory mechanisms are unclear. To investigate the in
vivo regulation of human preproendothelin-1 (PPET-1), we examined the
activity of the PPET-1 promoter in transgenic mice exposed to hypoxia.
Mice expressing one of three PPET-1 promoter-luciferase (PPET-1/LUC)
reporter transgenes (2.5 kb, 138 bp, or none of the
5'-flanking sequences of the PPET-1 gene) were generated. LUC
expression was reduced in mice with a truncated 138-bp PPET-1 promoter.
Exposure of mice bearing the 2.5-kb PPET-1/LUC transgene to hypoxia
(10% O2 for 24 h) increased LUC
expression sixfold in pulmonary tissue but only twofold in other
tissues. In situ hybridization revealed the strongest transgene
expression in the pulmonary vasculature and bronchiolar epithelium.
These data are consistent with the hypothesis that hypoxic induction of
the PPET-1 gene leads to increased pulmonary production of ET-1 in
diseases associated with low O2
tension.
endothelin; heart failure; pulmonary hypertension
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INTRODUCTION |
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THE ENDOTHELIN (ET) peptides (ET-1, ET-2, and ET-3) comprise a family of structurally and pharmacologically related agents with potent biological activity, including contraction and vasodilation of smooth muscle, pressor and depressor effects in the renal vasculature, and positive myocardial inotropy and chronotropy (47). These biologically active peptides arise from specific preproendothelin (PPET) proteins, each of which is encoded by a separate gene. ET-1 was originally isolated from porcine endothelial cells (46), but it has since been shown to be produced by vascular smooth muscle cells (37), respiratory epithelial cells (38), renal glomerular, epithelial and mesangial cells (6, 19), keratinocytes (48), and neurites (26, 30). Significant elevations in plasma ET-1 accompany pulmonary hypertension (10), congestive heart failure (CHF) (4, 21, 27), cardiogenic shock (5), systemic hypertension (20), and myocardial ischemia (32, 35, 43), suggesting that ET-1 production and/or hydrolysis are altered in these conditions. ET receptors are also affected by ischemic disease. Nambi et al. (34) observed an altered affinity of ET receptors for 125I-labeled ET-1 after renal ischemia, and Bird et al. (2) reported that ETA receptor density is increased in kidneys from spontaneous hypertensive rats compared with normotensive rats. The actions, associations, and responsiveness of the components of the ET receptor system to physiological and pathophysiological stimuli has led to the suggestion that ET may be causally involved in a variety of disease processes.
Attempts to define a role for ET in disease have employed various inhibitors of ET action, including receptor antagonists, anti-ET antibodies, and inhibitors of endothelin-converting enzyme. Several antagonists have significantly attenuated neointima formation after balloon injury of the rat carotid artery (7, 9). Kowala et al. (23) demonstrated that the ETA subtype-selective antagonist BMS-182874 decreased atherosclerotic lesion size in hamsters fed high-cholesterol diets compared with vehicle-treated control animals. Balanced ET receptor antagonism with bosentan had beneficial effects on hemodynamic and cardiac parameters in patients with class III heart failure (18). In ischemic myocardial disease, anti-ET-1 antibodies (43), phosphoramidon, a nonspecific endothelin-converting enzyme inhibitor, and ETA receptor antagonists (13, 14) protected ischemic hearts in vivo. These data are consistent with an emerging role for ET in a variety of vascular diseases.
Another approach to elucidation of the role of ET in disease is through transgenic animals in which the PPET genes are either overexpressed or ablated through homologous recombination. Recent work with mice rendered deficient in ET-1 through homologous recombination has shown that ET-1 is essential for normal development and cardiovascular homeostasis (24). Puffenberger and colleagues (36) have also shown that a missense mutation in the ETB receptor gene is involved in Hirschsprung's disease, a syndrome characterized by altered innervation of the distal colon. Moreover, targeted or natural disruption of the ETB gene (17) or targeted disruption of the ET-3 gene (12) mimics Hirschsprung's disease. In contrast, no successful attempts to overexpress the PPET-1 gene have been reported.
Despite significant elevations in circulating and tissue levels of ET in diseases of low O2 tension, such as pulmonary hypertension and CHF, the regulatory mechanisms underlying disease-associated increases in ET-1 production remain unclear. Studies have shown that PPET-1 mRNA and protein levels increase in cultured human endothelial cells exposed to hypoxia (22) and in pulmonary vascular endothelial cells of patients with primary pulmonary hypertension (10). Studies in rats have shown that exposure to hypoxia increases PPET-1 mRNA levels in lungs and pulmonary arteries (8, 28). To begin to elucidate the in vivo regulation of human PPET-1 gene expression in pulmonary hypertension and CHF, transgenic mice that express the firefly luciferase (LUC) reporter gene under the control of the human PPET-1 promoter were developed. In the present study, we show that pulmonary-specific enhancement of the human PPET-1 promoter activity occurs in vivo in response to hypoxia.
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MATERIALS AND METHODS |
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Subcloning and purification of the PPET-1/LUC constructs.
A 18-kb genomic fragment containing the full-length human PPET-1 gene
was generously provided by Dr. Tom Quertermous (Vanderbilt University;
see Ref. 3). For construction of a plasmid containing 2.459 kb of
5'-flanking sequences, a 2.6-kb
Xho
I/Bgl II PPET-1 fragment was subcloned
into the Xho I/BamH I
site of the LUC expression vector KSbLUC. The resulting plasmid,
PPET-1/LUC(2459), contains several
cis-acting regulatory elements
upstream from the transcription initiation site (Fig.
1). A second plasmid, containing only 138 bp of 5'-flanking sequence PPET-1/LUC(
138), was created by
subcloning a 308-bp Bsg
I/Bgl II fragment into the
Sma
I/Bgl II sites of KSbLUC expression
vector. This construct removes most of the 5' regulatory elements
but retains the GATA site as well as the TATA and CAAT boxes. For
transfection experiments, DNA was purified from agarose gels using the
Qiagen Plasmid Purification Kit, according to the manufacturer's
instructions (Qiagen, Chatsworth, CA).
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In vitro expression of PPET/LUC DNAs.
Before injection into oocytes, plasmid DNA containing fusion gene
constructs was transfected into bovine pulmonary artery endothelial
(BPAE) cells (American Type Culture Collection, Rockville, MD).
Confluent flasks of BPAE cells were seeded into 35-mm
plates and were allowed to incubate overnight to 50% confluence. Cells were then transfected with 3 µg of PPET-1/LUC(
2459),
PPET-1/LUC(
138), and KSbLUC DNA for 18 h using Lipofectin
reagent, according to the manufacturer's instructions (Life
Technologies, Gaithersburg, MD). Mock transfected cells incubated with
Lipofectin reagent without DNA and untreated cells were also tested.
After 48 h, cells were rinsed with phosphate-buffered saline (PBS) and
were lysed in 1× reporter lysis buffer (Promega, Madison, WI).
Lysates were centrifuged at 13,000 revolutions/min (rpm) for 30 s.
Supernatants were transferred to new tubes and either assayed
immediately or stored at
80°C.
Production and characterization of transgenic mice.
The PPET-1 promoter/LUC constructs, including SV40 polyadenylation
sequences PPET-1/LUC(2459), PPET-1/LUC(
138), and LUC, were excised from the KSbLUC vector and were purified by sucrose gradient centrifugation. The purified DNA constructs were microinjected into the male pronucleus of fertilized mouse eggs (C57BL/6J × DBA/2F2; Jackson Laboratory, Bar
Harbor, ME). Microinjected eggs were then transferred into the oviducts
of Institute for Cancer Research foster mothers (Harlan
Sprague Dawley, Indianapolis, IN). Identification of transgene-positive
mice was conducted by polymerase chain reaction (PCR) analysis of mouse
genomic DNA with two sets of primers: one that amplifies a 359-bp
fragment of the firefly LUC gene and a second set that amplifies a
490-bp fragment of the endogenous ZP3
control gene (29).
RNA analysis.
Mouse tissues were excised, immediately frozen in liquid
N2, and stored at 80°C
until processing. Total RNA was isolated using the UltraSpec RNA
Isolation System, according to the manufacturer's instructions
(Biotecx, Houston, TX). Precipitated RNA was resuspended and stored in
formamide. RNA yield was quantitated by spectrophotometry and was
confirmed by agarose gel electrophoresis with ethidium bromide
staining. Thirty and three micrograms of RNA were applied to a Nytran
membrane (Schleicher & Schuell, Keene, NH) by vacuum blotting using a
slot-blot manifold (Hoefer Scientific, San Francisco, CA).
Hybridization was performed in 50% formamide, 5× sodium
chloride-sodium phosphate-EDTA (SSPE), 1×
Denhardt's solution, 1% sodium dodecyl sulfate (SDS), and 100 µg/ml
sheared herring testes DNA at 42°C. A 559-bp probe that hybridizes
to a sequence in the 3'-untranslated region of the PPET-1 mRNA
was labeled by random priming using the RediPrime System, according to
the manufacturer's instructions (Amersham, Arlington Heights, IL), and
was added to the hybridization solution. After overnight incubation,
the slot blot was washed two times with 2× SSPE and 0.1% SDS at
42°C and two times with 0.5 SSPE and 0.1% SDS at 60°C. The
relative intensity of each slot was quantitated using a PhosphorImager
(Molecular Devices, Sunnyvale, CA), and data quantitation was conducted
with ImageQuant image analysis software.
LUC assays of cell and tissue extracts.
Organs excised from mice were immediately frozen in liquid
N2 and were then sonicated in a
solution containing 25 mM glycylglycine (pH 7.8), 15 mM
MgSO4, 1 mM dithiothreitol (DTT),
and 1% Triton X-100. Lysates were centrifuged at 12,000 rpm in a
microfuge for 30 min at 4°C. Supernatants were transferred to
another tube and were stored at 80°C until assay. Extracts
from cells and tissues were assayed using the Luciferase Assay System,
according to the manufacturer's instructions (Promega). Ten (cells) or
twenty-five (tissues) microliters of lysate were injected with one
hundred microliters of luciferase assay reagent [20 mM Tricine,
1.07 mM (MgCO3)4Mg(OH)2-5H2O,
0.1 mM EDTA, 33.3 mM DTT, 270 µM CoA, 470 µM luciferin,
and 530 µM ATP]. Light emissions were quantitated for 30 s at
25°C in a luminometer (EG & G Berthold Automat LB 953, Princeton,
NJ).
Hypoxia treatment. To investigate the activity of the PPET-1 promoter under low O2 conditions, the effect of hypoxia on transgene expression was examined. Mice at 8-10 wk of age were exposed for 24 h to room air or normobaric hypoxic (10 ± 0.5% O2) conditions in a box in which CO2 was kept at <0.2% and relative humidity was kept at <70% as previously described (28). Immediately after the 24-h exposure period, tissues were harvested for LUC assay.
In situ hybridization.
At the end of the 24-h hypoxic exposure, mice were removed individually
from the hypoxic chamber or from their cages and were rapidly killed by
decapitation. The lungs were excised quickly, rinsed in cold PBS
solution, and fixed in 4% paraformaldehyde for 3 h and in 15% sucrose
overnight at 4°C. Frozen samples were embedded in HistoPrep frozen
tissue-embedding media (Fisher Scientific, Pittsburgh, PA) on dry ice
and were stored at 80°C.
Statistical analysis. Statistical significance was determined using analysis of variance or two-tailed Student's t-test. Significant F-values were evaluated with a pairwise test of individual group means using Bonferroni probabilities.
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RESULTS |
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In vitro expression of PPET-1/LUC DNAs.
To determine if the PPET-1/LUC(2459) and PPET-1/LUC(
138)
DNAs were expressed in vitro, BPAE cells were transiently transfected with plasmid DNA containing these constructs. Comparison of LUC activities in the PPET-1/LUC(
2459) and PPET-1/LUC(
138)
transfected cells demonstrates that, while both constructs confer LUC
expression on otherwise null cells, the truncated promoter drives LUC
expression at an approximately eightfold lower levels than does the
2.5-kb promoter and 5'-flanking sequences. Cells transfected with
the KSbLUC vector had negligible LUC activity (Fig.
2). Equivalent transfection efficiencies
were confirmed by cotransfection in the presence of a
-galactosidase
reporter vector (data not shown). This finding is consistent with the
suggestion that regulatory elements in the 5'-sequence upstream
of
138 bp facilitate PPET-1 expression.
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In vivo expression of PPET-1/LUC DNAs.
After excision of the transgenes from the KSbLUC vector, transgenic
founders were generated expressing the PPET-1/LUC(2459), PPET-1/LUC(
138), and LUC constructs (Fig. 1). Of the animals tested, 10 positives were identified for PPET-1/LUC(
2459). In addition, 10 positives were identified for PPET-1/LUC(
138). The PPET-1/LUC(
138) mice were used for validation of promoter
expression. In addition, eight positives were identified for LUC (data
not shown).
Validation of PPET-1 promoter activity.
To assess if the PPET-1/LUC(2459) transgene was expressed in a
physiologically relevant manner, the tissue pattern of LUC activity in
PPET-1/LUC(
2459) transgenic mice was compared with endogenous
PPET-1 mRNA expression in normal mice. PPET-1 mRNA was detected in all
tissues examined, and the presence of PPET-1 mRNA correlated with the
presence of LUC enzyme activity (Table 1).
These data demonstrate that the PPET-1/LUC(
2459) transgene is
expressed in the same tissues as endogenous ET-1.
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Effect of hypoxia on PPET-1 promoter activity.
LUC activity was assessed in PPET-1/LUC(2459) mice exposed for
24 h to room air or hypoxic (10%
O2) conditions. Hypoxia
significantly stimulated LUC enzyme activity in the lung, heart,
kidney, liver, and spleen (Fig. 4A).
The greatest induction was observed in pulmonary tissue, in which
hypoxia stimulated a sixfold increase in LUC over room air controls.
Hypoxic induction of LUC was twofold over control in heart, kidney,
liver, and spleen. There was no stimulation of LUC activity in the tail
(data not shown). Verification that hypoxic induction of LUC activity
is mediated via the PPET-1 promoter was demonstrated by the absence of
LUC activity in promoterless LUC mice after exposure to 10%
O2 for 24 h (Fig.
4B).
These data demonstrate that PPET-1 promoter activity is stimulated by
hypoxia in a pulmonary-specific manner.
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Cellular localization of PPET-1/LUC transgene expression.
The cellular localization of PPET-1/LUC(2459) expression in lung
from hypoxia-exposed transgenic mice was determined using in situ
hybridization with an antisense LUC probe. In lungs of hypoxic mice,
strong hybridization was detected in pulmonary vessels in both smooth
muscle and endothelial cells (Fig.
5A).
Expression of the transgene was not confined to vascular tissue, as
antisense hybridization was also detected in pulmonary bronchiolar
epithelial cells and alveoli (Fig.
5A). Diffuse hybridization was also
observed in the lung parenchyma. The hybridization patterns were
similar, but substantially lower, in normoxic mice (data not shown).
Hybridization of the sense LUC probe to hypoxic and normoxic mice
yielded diffuse and nonspecific staining (Fig.
5C).
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DISCUSSION |
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To elucidate the in vivo regulation of human PPET-1 gene expression in pulmonary hypertension and CHF, transgenic mice that express the firefly LUC reporter gene under the control of the human PPET-1 promoter were developed. The dynamic range of the LUC reporter against a null background in mice affords a much more sensitive in vivo assay to assess promoter activity than measuring PPET-1 mRNA. In the present study, we show that pulmonary-specific enhancement of the human PPET-1 promoter activity occurs in vivo in response to hypoxia.
Several lines of evidence demonstrate that the PPET-1/LUC(2459)
transgene is expressed in a physiologically relevant manner. First, the
tissue pattern of PPET-1 transgene expression is consistent with the
endogenous mouse PPET-1 mRNA expression pattern observed here and
reported elsewhere. Sakurai et al. (40) studied endogenous PPET-1
expression in the rat and found strong hybridization in heart, lung,
kidney, spleen, submandibular gland, liver, and stomach. Hilkert et al.
(16) studied endogenous PPET-1 mRNA expression in mice and determined
that lung, brain, kidney, spleen, and heart were positive tissues.
Additional studies have since demonstrated the presence of ET-1
transcripts in bladder (39) and human skin (48). Thus the data reported
here for expression of the PPET-1/LUC transgene are consistent with
previous observations of the endogenous expression of PPET-1 mRNA.
A second line of evidence in support of the physiological regulation of
the PPET-1/LUC(2459) transgene derives from studies with a
truncated promoter. Wilson and colleagues (45) previously observed that
deletion of the 5'-flanking sequence of human PPET-1 affected
expression of a growth hormone (GH) reporter gene. Constructs containing the proximal 157 bp of the PPET-1 promoter exhibited near
maximal expression of GH, whereas truncations beyond
157 bp
significantly attenuated GH production. We observed an eightfold decrease in promoter activity in cells transfected with a promoter truncated at
138 bp. Similarly, in vivo expression of the
PPET-1/LUC transgene with only 138 bp of 5'-flanking sequence is
10- to 30-fold reduced in most tissues. The observation that expression
of PPET-1/LUC(
2459) and PPET-1/LUC(
138) was the same in
aorta suggests that additional cis-acting regulatory elements not
present in the 2.5-kb upstream region of the PPET-1 gene are required
for full expression in this tissue. Regulatory elements present in the
5'-region include a consensus sequence for an acute phase
regulatory element thought to mediate the induction of mRNA under acute
physical stress, two activator protein (AP)-1/JUN- or
12-O-tetradecanoylphorbol-13-acetate-responsive elements
known to modulate transcriptional activation in many mammalian systems,
and a nuclear factor-1 consensus sequence that mediates
transcriptional activation by transforming growth factor-
. Alternatively, these tissues may not express the complement of transcription factors that interact with the region of the
5'-flanking sequence spanning
138 to
2459 bp.
In situ hybridization with specific antisense LUC and mouse PPET-1
probes in hypoxic PPET/LUC(2459) mice demonstrates
colocalization of these mRNAs. We observed expression of LUC and PPET-1
mRNA in pulmonary vessels, bronchiolar epithelium, and alveoli.
Interestingly, although high levels of LUC expression were detectable
in both the endothelium and smooth muscle of pulmonary vessels, PPET-1 expression was mostly in the endothelium. This difference in cellular localization could be due to the absence of additional
cis-acting elements in the
PPET/LUC(
2459) construct that may confer cell specificity. Our
data are consistent with previous studies that examined the patterns of
PPET-LUC transgene expression in mice. Immunostaining with anti-LUC
antibodies has been previously observed in murine bronchiolar
epithelium (15). These investigators observed that a 2.4-kb segment of
5'-flanking sequence, the first exon of the gene and 0.8 kb of
the first intron, directed LUC transgene expression to the endothelium
of large and small vessels as well as to arterial smooth muscle and
select epithelial surfaces. Using a 4.3-kb segment of the human PPET-1
5'-flanking and promoter sequence, Hilkert et al. (16)
demonstrated a cell-specific expression pattern of the transgene in
endothelial cells of blood vessels and epithelial cells of the lung.
Significant elevations in circulating and tissue ET levels accompany
numerous cardiovascular diseases. The present data suggest that the
2.5-kb 5'-flanking sequence of the human PPET-1 gene contains a
hypoxia-responsive element that is activated in a pulmonary-specific manner. These findings are consistent with the suggestion that in
diseases associated with low O2
tension, such as pulmonary hypertension and CHF, hypoxic activation of
the PPET-1 gene leads to increased pulmonary production of ET-1. The
responsiveness of the human PPET-1/LUC(2459) transgene to
hypoxia is consistent with previous accounts of increased plasma levels
of ET-1 and steady-state PPET-1 mRNA levels after exposure to hypoxia.
Acute (24-48 h) or chronic (4 wk) exposure of rats to hypoxic
conditions elevated plasma ET-1 and also caused a selective increase in
pulmonary PPET-1 mRNA levels as little or no increase in PPET-1 mRNA
was observed in organs perfused by systemic circulation (8, 28, 42).
Thus the present observation of greater induction of LUC activity in
lung compared with other tissues confirms and extends these earlier
observations. In humans, plasma ET-1 levels increase progressively with
the hypoxia associated with high altitude (33). This increase in plasma
levels is likely the result of increased PPET-1 gene transcription as
Kourembanas and colleagues (22) showed that hypoxia increases PPET-1
gene transcription rates in cultured human endothelial cells. Taken
together, these data suggest that the elevations in circulating ET-1
observed in hypoxia are due primarily to increased pulmonary PPET-1
gene transcription.
The mechanism for the hypoxic induction of the PPET-1 gene is unclear.
Previous studies with hypoxia-sensitive genes such as erythropoietin
(EPO) and vascular endothelial growth factor (VEGF) (1, 11, 25, 31)
suggest that a hypoxia-responsive enhancer (HRE) and hypoxia-inducible
factor-1 (HIF-1) are involved. Analysis of the 5'-flanking
sequence of the PPET-1 gene shows that a 7-bp-long motif similar to the
core HIF-1 binding site of the EPO and VEGF genes (41) exists at
position 117 to
124. We speculate that the PPET-1 gene
belongs to the set of hypoxia-inducible genes and that the lung
contains greater quantities of HIF-1 available for interaction with the
HRE in PPET-1 than other organs such as heart or kidney. Additional
studies are necessary to test this hypothesis and to determine if
hyperoxia has a similar or different effect on PPET-1 transcription.
In summary, the data presented here provide evidence for pulmonary-specific hypoxic induction of the PPET-1 promoter and support the hypothesis that pulmonary production of ET-1 contributes to diseases associated with low O2 tension such as pulmonary hypertension and CHF. The PPET-1/LUC transgenic mice described here will be useful in delineating this mechanism and role of ET-1 production during the course of specific disease progression.
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ACKNOWLEDGEMENTS |
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We extend thanks to Dr. Koji Maemura of the University of Tokyo for the murine PPET-1 gene. We also gratefully acknowledge the expertise of Petronio Zalamea for oocyte injection and thank the Veterinary Science Department of Bristol-Myers Squibb and the University of Alabama for scientific expertise and animal care during the course of these studies.
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FOOTNOTES |
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Address for reprint requests: M. L. Webb, Pharmacopeia Inc., 3000 Eastpark Blvd, Cranbury, NJ 08512.
Received 4 February 1997; accepted in final form 10 July 1997.
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