Departments of 1 Pediatrics and 4 Molecular Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611-3008; and 2 Department of Pediatrics and 3 Cardiovascular Research Institute, University of California, San Francisco, California 94143-0106
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ABSTRACT |
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Endothelial nitric oxide synthase (eNOS) mRNA and protein levels increase during late gestation and then decrease postnatally in sheep lung parenchyma. The increase in fluid shear stress at birth, resulting from increased pulmonary blood flow, is an important mediator of postnatal eNOS gene expression. Our objective was to identify factors stimulating eNOS expression in pulmonary arterial endothelial cells (PAEC) in response to shear stress and to determine if these factors are developmentally regulated. PAEC were isolated from fetal lambs and adult sheep. Transcriptional activity from a 1,600-bp eNOS promoter fragment increased in both fetal and adult PAEC exposed to 8 h of shear stress. Conversely, activity driven from an 840-bp promoter fragment containing a putative activator protein (AP)-1 binding site was increased only in fetal PAEC. This increase was completely abolished in an identical construct containing a mutant AP-1 sequence. The AP-1 protein c-Jun was localized to the cytosol in static adult PAEC and to the nucleus in static fetal PAEC. After shear, c-Jun was nuclear localized in both cell types. However, transcriptionally active phosphorylated c-Jun was elevated only in the nuclei of sheared fetal PAEC. Resting levels of eNOS and NO were 2- and 20-fold higher, respectively, in fetal cells. Shear increased eNOS and NO in both cell types: levels were ~2.5-fold higher in fetal PAEC. Phosphorylation of Akt and eNOS was evident in sheared fetal but not adult PAEC. We have therefore identified mechanisms of eNOS regulation at the transcriptional level and to be enzyme activation specific to the fetal pulmonary arterial circulation.
activator protein-1; nitric oxide; fetal sheep; pulmonary arterial endothelial cells; gene expression; maturational differences; protein translocation
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INTRODUCTION |
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AFTER BIRTH, WITH INITIATION of ventilation of the lungs, pulmonary vascular resistance decreases and pulmonary blood flow increases 8- to 10-fold to match systemic blood flow (9, 13, 28, 52). This process is regulated by a complex and incompletely understood interplay between mechanic and metabolic factors (18). Recent evidence suggests that normal pulmonary vascular tone is regulated by a complex interaction of vasoactive substances produced by the vascular endothelium including nitric oxide (NO) (18, 22, 27, 60). NO is an endothelium-derived relaxing factor synthesized by the oxidation of L-arginine after activation of endothelial NO synthase (eNOS) (46). L-Arginine increases pulmonary blood flow in fetal and newborn lambs and augments endothelium-dependent pulmonary vasodilation (1, 17). In addition, inhaled NO decreases pulmonary vascular resistance in fetal lambs and in newborn lambs with pulmonary hypertension (1, 32, 61). Conversely, inhibition of NO synthesis increases pulmonary vascular resistance in fetal lambs (1, 12).
Several studies have investigated the potential role of NO in the
developing pulmonary circulation. Both basal and agonist-stimulated endothelial production of NO has been demonstrated in the fetal, newborn, and adult vasculature. Basal NO production rises twofold from
late gestation to 1 wk of life and another 1.6-fold from 1 to 4 wk of
life in intrapulmonary arteries (55). A physiological study of intrapulmonary arteries and isolated lung preparations from
sheep revealed maturational increases in NO-mediated relaxation during
the late fetal and early postnatal periods (55).
Similarly, in the pig, there is an increase in NO-mediated relaxation
during the first 2 wk of life, followed by a decrease (37,
48). Coinciding with these data, eNOS mRNA and protein increase
in late gestation then decrease postnatally in rat and sheep lung
parenchyma (26, 30, 45). In addition, lung mRNA levels for
the 1-and
1-subunits of soluble guanylate
cyclase during late-gestation fetal and neonatal rats are sevenfold
higher than those found in adult rats (7). This
maturational increase in NO and cGMP production parallels the dramatic
decrease in pulmonary vascular resistance that occurs at birth. Also,
in the late-gestation fetal lamb, an infusion of the eNOS inhibitor
N
-nitro-L-arginine markedly
attenuates the increase in pulmonary blood flow associated with
ventilation at birth (1, 17). Together, these data
strongly suggests that NO activity mediates, in part, the fall in
pulmonary vascular resistance during the transitional pulmonary
circulation and maintains the normal low postnatal pulmonary vascular
resistance. The developmental regulation of eNOS gene expression is
likely to play a key role in controlling the fetal to newborn transition.
eNOS gene expression is influenced by several stimuli, with laminar shear stress making an important contribution. Fluid shear stress is defined as the tractive force produced by moving a viscous fluid (blood) on a solid body (vessel wall), constraining its motion (41). When endothelial cells (EC) are subjected to shear stress, diverse responses are initiated, some of which occur within minutes and others that develop over several hours or days (42). NOS activity, NO production, and eNOS mRNA and protein levels are increased in EC exposed to shear stress (33-35, 43, 44, 49, 57, 58). Furthermore, studies have shown that the increased pulmonary blood flow occurring at birth leads to an increase in eNOS expression in the pulmonary vascular endothelium of newborn lambs (5, 19).
To identify the factors involved in this pathway leading from increased pulmonary blood flow to increased eNOS expression, we have previously exposed fetal pulmonary arterial endothelial cells (FPAEC) to laminar shear stress. NO production was found to be biphasic with an increase in NO up to 1 h, a plateau phase until 4 h, then a second release between 4 and 8 h (59). The initial release may have been due to the activation of preformed enzyme. In contrast, the second NO release coincided with an increase in eNOS mRNA and protein and may have been stimulated by factors that regulate eNOS gene transcription. PKC isoforms appear to be important in both stages of NO release, although our data suggest that the two pathways are distinct (59).
The purpose of this study was to identify additional components of the shear stress-induced signaling pathway. To identify which of these components are potentially developmentally regulated, we also determined whether the same mechanisms activate eNOS expression in adult PAEC (AdPAEC). Here we demonstrate the involvement of activator protein (AP)-1 in the activation of eNOS transcription in FPAEC, but not in AdPAEC, exposed to laminar shear. In addition, shear stress-induced pathways that mediate the phosphorylation of Akt, eNOS, and c-Jun are present only in FPAEC.
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MATERIALS AND METHODS |
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Animals. All procedures and protocols were approved by the Committee on Animal Research of the University of California at San Francisco. Five mixed-breed Western ewes at each maturational age [118-120 days (d), 138-140 d of gestation, newborn (<2 d), and adult] were euthanized with an intravenous injection of pentobarbital sodium (Euthanasia CII; Central City Medical, Union City, CA) and were subjected to bilateral thoracotomy. These animals had not undergone previous surgery or study.
Tissue preparation.
The heart and lungs were removed en bloc. To prepare total RNA, lung
tissue was removed, snap-frozen in liquid nitrogen, and stored at
70°C until used. Total RNA was prepared from snap-frozen lung
tissue (after pulverization) or from cultured ovine pulmonary arterial
EC (see below) by brief homogenization in 4 M guanidinium isothiocyanate, followed by extraction with acid phenol and
precipitation in isopropanol (11). To prepare protein,
lung tissue was rinsed in cold (4°C) normal saline to remove blood,
then minced, and finally homogenized using a Tissuemizer (2 × 15 s at 80% power) at 4 vol/wet wt of Triton lysis buffer (20 mM
Tris · HCl, pH 7.6, 0.5% Triton X-100, and 20%
glycerol) supplemented with protease inhibitors; cultured ovine
pulmonary arterial EC were sonicated in 3 vol/wet wt. Extracts were
then centrifuged at 15,000 g for 15 min. The supernatant was
then removed for protein determination and Western blot analysis
(29, 30).
Cell culture techniques. The heart and lungs were obtained from fetal lambs (138- to 140-d gestation), newborn lambs (<2 d), or adult sheep after death. These animals had not undergone previous surgery or study. The main and branch pulmonary arteries were removed and dissected free; the adventitia was removed. The exterior of the vessel was rinsed with 70% ethanol. The vessel was then opened longitudinally, and the interior was rinsed with PBS to remove any blood. Using a cell scraper, we lightly scraped away the endothelium, placed it in medium DMEM-H16 (with 10% fetal bovine serum and antibiotics), and incubated it at 37°C in 21% O2-5% CO2 balanced with N2. After 5 days, islands of EC were cloned to ensure purity. Basic fibroblast growth factor (1 ng/ml, a gift from Dr. Denis Gospodarowicz, Chiron, Emeryville, CA) was added to the media every other day. When confluent, the cells were passaged to maintain them in culture or frozen in liquid nitrogen. EC identity was confirmed by their typical cobblestone appearance, contact inhibition, specific uptake of 1,1'-dioctadecyl-1,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (Molecular Probes, Eugene, OR), and positive staining for von Willebrand factor (Dako, Carpinteria, CA). Ovine pulmonary arterial EC were studied between passages 3 and 10.
Generation and transfection of promoter constructs.
We generated promoter constructs by PCR using human genomic DNA as a
template. The reverse primer common to all three constructs binds
immediately upstream of the ATG initiation codon of eNOS (5'-GTTACTGTGCGTCCACTCTGCTGCC). Forward primers were:
5'-ATCTGATGCTGCCTGTCACCTTGAC (1,600-bp fragment),
5'-TGT-AGTTTCCCTAGTCCCCC (840-bp fragment), and
5'-GGTGTGGGGGTTCCA-GGAAA (650-bp fragment). A 2-bp mutation was
introduced into the AP-1 consensus sequence within the 840-bp construct. The sequence TGAGTCA at 661 in the human eNOS promoter (39) was changed to TGAGTtg by site-directed mutagenesis
(Retrogen, San Diego, CA) based on previous studies (8,
29). All promoter constructs were sequenced to confirm their
identity. Fetal and adult EC were transfected with 10 µg of plasmid
DNA on a 10-cm2 tissue culture plate at 90% confluence
with Lipofectamine (GIBCO-BRL) according to the manufacturer's
instructions. After 48 h, the two cell types were split onto three
wells of two six-well plates and allowed to adhere for a further
24 h. Medium was replaced with serum-free DMEM, and cells were
left static or were subjected to laminar shear stress (for 8 h) as
described below, such that sheared cells and static controls came from
the same transfection reaction. We determined luciferase activity of 10 µl of protein extracts using the Dual-Luciferase Reporter Assay
System (Promega) and a Femtomaster FB12 luminometer (Zylux).
Fluid shear stress.
A cone-plate viscometer similar to that described by Sdogous et al.
(54) was designed and built such that it accepts six-well tissue culture plates. This apparatus consists of a cone of shallow angle () rotating at angular velocity (
) on top of a tissue culture plate containing medium of a known viscosity (µ) on which EC
were present as a monolayer. This allowed the monolayer to be subjected
to a radially constant fluid-shear stress (
, dyn/cm2) as
calculated with the formula
=
/µ. A cone angle of
0.5° is used to achieve laminar flow rates representing levels of
shear stress within physiological parameters. Typical physiological shear stress in the major human arteries is in the range of 5-20 dyn/cm2 (38) with localized increases to
30-100 dyn/cm2 (15). Thus we imparted a
shear stress of 20 dyn/cm2 to mimic the upper limit of the
physiological range. Also, since previous studies have shown that
pulsatile and laminar flow appears to induce similar effects on NO
production, laminar shear stress was employed (44).
RNase protection assay. The plasmid containing the ovine eNOS cDNA fragment was linearized with the appropriate restriction enzyme (GIBCO-BRL, Grand Island, NY). We synthesized antisense single-stranded ovine eNOS riboprobes by in vitro transcription using T3 RNA polymerase (Boehringer-Mannheim, Indianapolis, IN) in the presence of cold rCTP, rGTP, and rATP. RNA probes labeled with [32P]UTP (New England Nuclear, Boston, MA) were used for RNase protection assays (4-6). RNase protection assays were performed as previously described (4-6). We did studies on total RNA prepared from peripheral lung tissue, using the antisense radiolabeled cRNA eNOS probe. Antisense radiolabeled single-stranded ovine RNA probes were hybridized overnight at 42°C with total RNA isolated peripheral lung (50 µg) in 80% formamide, 50 mM PIPES (pH 6.4), 0.4 M NaCl, and 1 mM EDTA. Single-stranded RNA was digested with an RNase A-T1 mixture (Ambion, Austin, TX) for 1 h at 37°C. After phenol-CHCl3 extraction and ethanol precipitation, the protected fragments were analyzed by electrophoresis on a 6% denaturing polyacrylamide gel. Also included was a probe for 18S to serve as a control for the amount of input total RNA and the recovery of protected probe fragments. In all experiments, sufficient counts were added such that the eNOS and 18S riboprobes were always in molar excess.
Western blot analysis. Western blot analysis was performed as previously described (4, 5, 59). Briefly, protein extracts (20 µg) were separated on 7.5% denaturing polyacrylamide gels. All gels were electrophoretically transferred to Hybond-polyvinylidene difluoride membranes (Amersham, Arlington Heights, IL). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween. After being blocked, the membranes were incubated at room temperature with a specific monoclonal antiserum raised against eNOS (1:2,500; Transduction Laboratories) then incubated with a goat anti-mouse IgG-horseradish peroxidase conjugate. After washing, chemiluminescence was used to detect the protein bands.
Immunocytochemistry and fluorescence detection. Sheared and static cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Washington, PA) for 20 min at 4°C. After three washes in phosphate-buffered saline (PBS; Cellgro), cells were permeabilized in 0.1% Igepal CA-630 (Sigma) for 1 min at room temperature and washed three more times in PBS. Cells were blocked in 5% (wt/vol) nonfat dry milk (Challenge Dairy Products) and 0.05% Tween (Fisher Biotech) in PBS for 60 min at room temperature. Cells were washed three times in PBS and incubated with primary antibodies against c-Jun (1:100; Oncogene Research Products), phosphorylated c-Jun (serine 73, 1:1,000; Cell Signaling), eNOS (1:1,000, Transduction Laboratories), phosphorylated eNOS (serine 1,177, 1:1,000; Cell Signaling), Akt (1:100, Cell Signaling), and phosphorylated Akt (serine 473, 1:100; Cell Signaling) in 5% BSA (Sigma), 0.05% Tween in PBS at 4°C for 16 h. Cells were washed three times in PBS and incubated with goat anti-rabbit (c-Jun, phosphorylated c-Jun, phosphorylated eNOS, and Akt) or goat anti-mouse (eNOS and phosphorylated Akt) IgG conjugated to rhodamine red or Oregon green (all from Molecular Probes) in 5% nonfat dry milk, 0.05% Tween in PBS for 60 min in the dark at room temperature. Cells were washed three times in PBS and visualized by fluorescence microscopy with excitation at 518 nm and emission at 605 nm for rhodamine red and with excitation at 485 nm and emission at 530 nm for Oregon green. Cells were imaged under a Nikon Eclipse TE-300 fluorescent microscope. Fluorescent images were captured with a CoolSnap digital camera, and the average fluorescent intensities (to correct for differences in cell number) were quantified with Metamorph imaging software (Fryer). Statistical analyses between treatments were carried out as detailed below (see Statistical analysis).
Diaminofluorescein fluorescence. Cells were loaded with 10 µM 4,5-diaminofluorescein diacetate (DAF2-DA) and incubated at 37°C for 30 min. Cells were washed three times in PBS and visualized by fluorescence microscopy at 485 nm for excitation and 530 nm for emission. Images were captured and quantified as described above.
Statistical analysis. Relative luminescent intensity was determined for the four promoter-luciferase constructs in sheared AdPAEC and FPAEC and expressed as fold change (shear/static) ± SD. The relative fluorescent intensity was calculated for secondary antibodies and DAF2-DA, then expressed as fold change (vs. AdPAEC static) ± SD. We made comparisons between treatment groups by the unpaired t-test using the GB-STAT software program. A P < 0.05 was considered statistically significant.
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RESULTS |
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RNase protection assays were performed to determine eNOS gene
expression in peripheral lung tissue from fetal lambs [120-d gestational age (F120d) and F140d], newborns (<2 d old), and adult sheep. The expression of eNOS mRNA was found to be maximally expressed in the newborn (P < 0.05 vs. F120d) and then to be
significantly decreased in the adult (P < 0.05 vs.
F120d; Fig. 1, A and
B). To determine whether these maturational differences in
eNOS expression could be maintained in culture, we prepared EC from
F140d or newborn lambs or adult sheep (Fig. 1, C and
D). These cells were then passaged 10 times (1:4 split when
cultures obtained confluency). At passage 10, RNA was
prepared, and eNOS expression determined by RNase protection assay
(Fig. 1C). The results obtained indicate that the eNOS
developmental phenotype can be maintained during 10 passages such that
eNOS expression is highest in newborn PAEC and is less in PAEC obtained
from adult sheep (Fig. 1D). Similar results were obtained
when eNOS protein levels were examined (Fig. 5).
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We then wished to determine whether there are maturational differences
in the cis-elements used to regulate the eNOS promoter. Because we have shown previously that eNOS mRNA levels increase in
FPAEC exposed to 8 h of laminar shear (59), we used
this to examine differences between PAEC isolated from either fetal (FPAEC) or adult sheep (AdPAEC). Thus we transfected both FPAEC and
AdPAEC with eNOS promoter fragments fused to a luciferase reporter
gene. Using a promoter fragment containing 1.6 kb of DNA 5' of the eNOS
transcription start site, we found luciferase activity increased
1.64-fold in FPAEC and 1.77-fold in AdPAEC after 8 h of laminar
shear (P < 0.05 vs. static controls, Fig. 2). A construct containing 840 bp of the
eNOS 5'-flanking region gave a 1.95-fold increase in luciferase
activity in FPAEC (P < 0.05 vs. static controls) but
no significant increase in AdPAEC after 8 h of shear (Fig. 2).
Conversely, a third construct containing 650 bp of the eNOS 5'-flanking
region was unresponsive to shear in both cell types (Fig. 2).
Inspection of the eNOS promoter sequence (39) between
840 and
650 identified a potential binding site for AP-1 at
661.
Mutation of two base pairs within the consensus AP-1 binding sequence
specifically disrupts DNA binding (8, 29). Introduction of
the same mutations within the AP-1 sequence of the 840-bp construct
completely abolished the shear-induced increase in luciferase activity
in the fetal cells (Fig. 2).
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To confirm that the endogenous eNOS gene in FPAEC requires the AP-1
element to respond to shear, we carried out an experiment in which
FPAEC were exposed to laminar shear stress for 8 h in the presence
or absence of the AP-1 inhibitor curcumin (10 µM). In the absence of
curcumin, eNOS mRNA levels increased by 4.9-fold from baseline;
however, in the presence of curcumin this increase was abrogated (Fig.
3), indicating the importance of
AP-1-mediated signaling in regulating eNOS gene expression by laminar
shear stress in FPAEC.
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AP-1 complexes comprise c-Jun homodimers or c-Jun/c-Fos heterodimers
(50, 53). Immunocytochemistry using an anti-c-Jun antibody
revealed a nuclear localization in resting FPAEC and a cytosolic
localization in resting AdPAEC, with 2.6-fold higher fluorescence in
FPAEC (P < 0.05 FPAEC vs. AdPAEC; Fig.
4, A and C). NO
release by sheared FPAEC is biphasic, with an increase up to 1 h,
a plateau, then a second release between 4 and 8 h (59). We were therefore interested in determining
differences between fetal and adult cells after 1 and 8 h of
shear. After 1 h of shear, c-Jun became nuclearly localized in
adult cells with a 2.6-fold and a 1.5-fold increase in fluorescence in
adult and fetal cells, respectively (P < 0.05; Fig. 4,
A and C). By 8 h, fluorescence was increased
by eightfold relative to resting AdPAEC in both cell types
(P < 0.05; Fig. 4, A and C). The
same localization and similar increases in fluorescence in response to
shear were seen using an anti-c-Fos antibody (data not shown). Once
bound at its cognate promoter sequence, c-Jun is phosphorylated at
serine 63 and serine 73 by c-Jun NH2-terminal kinase (JNK), rendering it transcriptionally active (3, 14, 36, 56). Immunocytochemistry indicated a twofold increase in phosphorylated c-Jun at serine 73 in FPAEC exposed to 8 h of laminar shear
(P < 0.05 vs. FPAEC static controls; Fig. 4,
B and D). However, no increase in phosphorylated
c-Jun was detected in sheared AdPAEC (Fig. 4, B and
D). Although we found a 1.5-fold increase in FPAEC sheared
for only 1 h, it was not statistically significant (Fig. 4,
B and D).
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Western blots were then performed to identify differences in eNOS
protein levels in peripheral lung tissue from fetal lambs (F120d and
F140d), newborns (<2 d old), and adult sheep. The expression of eNOS
protein was found to be maximally expressed in the newborn (P < 0.05 vs. F120d) and then to be significantly
decreased in the adult (P < 0.05 vs. F120d; Fig. 5,
A and B). We then
looked for differences in eNOS protein levels in
cells in culture. Resting FPAEC had twofold greater eNOS protein than
resting AdPAEC, as determined by Western blot analysis
(P < 0.05 vs. FPAEC vs. AdPAEC static; Fig. 5,
C and D). After 1 h of shear, eNOS levels
were unchanged, but by 8 h they had increased by 2.2-fold in FPAEC and 2.1-fold in AdPAEC (P < 0.05; Fig. 5, E
and F). We also used immunocytochemistry to detect changes
in eNOS protein levels. Quantification of fluorescent images was in
good agreement with the Western data, indicating twofold greater eNOS
protein in resting FPAEC (P < 0.05; Fig.
6, A and C). No
changes were detected after 1 h of shear, but by 8 h there
was a 2.7-fold and 1.7-fold increase in FPAEC and in AdPAEC,
respectively (P < 0.05; Fig. 6, A and C). eNOS activity is increased following
AKT/PKB-mediated phosphorylation at serine 1,177 (16,
20). Immunocytochemistry identified a 2.9-fold increase in
phosphorylated eNOS in FPAEC exposed to 8 h of laminar shear
(P < 0.05 vs. static controls; Fig. 6, B
and D). However, no increase in eNOS phosphorylation at
serine 1,177 was detected in sheared AdPAEC (Fig. 6, B and
D).
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We then used immunocytochemistry to confirm a role for Akt in the
shear-induced phosphorylation of eNOS in FPAEC. Akt protein levels were
similar in static fetal and adult cells, with no changes detected after
1 h of shear (Fig. 7, A and
C). After 8 h, there was
a 2.4-fold increase in sheared fetal cells (P < 0.05 vs. static; Fig. 7, A and C) but no significant
increase in the sheared adult cells (Fig. 7, A and
C). Phosphorylated Akt levels were similar in static fetal
and adult cells, with no changes detected after 1 h of shear (Fig.
7, B and D). After 8 h of shear, there was an 8.1-fold increase in the fetal cells (P < 0.05 vs.
static; Fig. 7, B and D) but no significant
increase in the sheared adult cells (Fig. 7, B and
D).
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Using the NO-sensitive fluorescent dye DAF2-DA, we determined that
resting NO production was 20-fold higher in FPAEC relative to AdPAEC
(P < 0.05 FPAEC vs. AdPAEC; Fig.
8, A and B). After 1 h of shear, DAF2-DA fluorescence increased 2.4-fold and 8.0-fold in FPAEC and AdPAEC, respectively (P < 0.05 vs.
static; Fig. 8, A and B). By 8 h of shear,
DAF2-DA fluorescence had increased 2.7-fold and 20.4-fold in FPAEC and
AdPAEC, respectively (P < 0.05 vs. static; Fig. 8,
A and B). Thus the levels of both eNOS protein
and NO in AdPAEC sheared for 8 h were similar to those found in
resting FPAEC.
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DISCUSSION |
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In the fetus, pulmonary vascular resistance is high and pulmonary
blood flow is low. With the initiation of ventilation and oxygenation
at birth, pulmonary vascular resistance decreases and pulmonary blood
flow increases. These rapid changes in pulmonary blood flow and
resistance are then followed by more gradual changes that occur over
the next several hours. Increasing evidence suggests that the changes
in pulmonary vascular tone are mediated by NO, possibly in response to
increased shear stress on the pulmonary vascular endothelium (26,
30, 45). In a number of clinical conditions, the pulmonary
circulation fails to undergo the normal transition to postnatal life,
resulting in persistent pulmonary hypertension of the newborn (PPHN)
(2, 6, 40). A better understanding of the mechanisms that
regulate the pulmonary vascular changes at birth may lead to new
prevention of and improved treatment strategies for PPHN. However, the
factors that mediate these changes remain unclear. Our initial studies
indicated that there are maturational differences in expression of eNOS
in the sheep lung. Our data indicate that eNOS mRNA and protein
expression peaked in the perinatal period and was significantly lower
in the adult. This data confirm that previously published by Parker et
al. (47). However, of more interest was our finding that
these developmental differences in eNOS mRNA and protein expression
could be maintained for at least 10 passages in culture, such that eNOS
expression was highest in PAEC isolated from newborn lambs followed by
those obtained from late-gestation lambs (F136d-F140d), whereas
expression in PAEC isolated from adult sheep was ~30% of that of the
late-gestation animals. This maintenance of maturational phenotype then
enabled us to determine whether there are differences in the response of eNOS to relevant physiological stimuli. To examine this, we chose to
determine the effect of increased pulmonary blood flow, as we have
previously shown that this can increase eNOS gene expression in both
fetal lambs and in PAEC isolated from these same lambs (5,
59). To mimic the effects of increased blood flow, we used a
cone-plate viscometer that allowed us to expose the PAEC monolayer to a
controlled level of shear stress. We have demonstrated an increase in
NO production and eNOS mRNA in FPAEC using this technique
(59). Using a 1.6-kb eNOS promoter construct, we found an
increase in eNOS transcription in both AdPAEC and FPAEC exposed to
8 h of shear. Data from promoter deletion constructs indicate that
the shear-induced increase in eNOS transcription is mediated via
cis-elements located between 840 and
650 bp in fetal,
but not in adult, PAEC. This suggests that additional upstream
sequences are required for transcriptional activity in the adult cells. Interestingly, the eNOS promoter contains a cis-acting
regulatory element identical to the previously identified shear
stress-responsive element (SSRE) (51) located at
994 bp,
raising the possibility that shear stress-induced factors regulate eNOS
transcription in AdPAEC via this sequence. Indeed, studies have
indicated that the SSRE is involved in the shear stress-induced
upregulation of the PDGF-B gene via the binding of the NF-
B
trans-acting factor (31). Further studies will be required
to determine whether this same pathway is activated by shear stress in
AdPAEC to increase eNOS expression.
A potential AP-1 binding site is located at 667 bp in the eNOS
promoter (39). Mutation of two base pairs in the consensus AP-1 binding sequence, predicted to specifically disrupt AP-1 binding
(8, 29) completely abolished the shear-induced increase in
eNOS promoter activity in fetal cells. Immunocytochemistry revealed
developmental differences in the localization of the AP-1 protein
c-Jun, which was anuclear in static AdPAEC but nuclear in static FPAEC.
After 1 h of laminar shear, c-Jun was nuclearly localized in
AdPAEC. After 8 h of shear, levels of c-Jun were identical in
fetal and adult cells. Furthermore, AdPAEC sheared for 8 h had
levels of eNOS protein and NO comparable with levels in static FPAEC.
Thus the sheared adult cells adopt a static fetal cell phenotype with
respect to NO production and c-Jun localization. The nuclear
localization signal (NLS) of c-Jun has been identified and has been
shown to direct nuclear translocation of an unrelated protein
(10). Although c-Jun is thought to be constitutively nuclear localized, the nuclear localization of its viral homolog v-Jun
is cell cycle dependent. Regulation of this translocation was mapped to
a single amino acid substitution in the NLS of v-Jun (10).
The mechanisms that determine c-Jun localization in FPAEC and AdPAEC
are unclear. It is possible that a c-Jun-binding protein masks the NLS
in static AdPAEC rendering it cytosolic, in the same way that NF-
B
is maintained within the cytosol by I
B binding (23). It
seems likely that this putative protein would be downregulated in
sheared AdPAEC and in FPAEC. Alternatively, NO-mediated pathways may
stimulate nuclear translocation of c-Jun, since sheared AdPAEC and
static FPAEC have similar NO levels. However, further studies will be
required to determine the mechanisms involved.
Although c-Jun becomes nuclearly localized in sheared AdPAEC, it does not appear to be phosphorylated and is therefore unlikely to be transcriptionally active. This may explain why the AP-1 promoter construct was insufficient to drive shear-stress-dependent luciferase transcription in the adult cells. Phosphorylation and activation of c-Jun are mediated by JNK (3, 14, 36). The reactive oxygen species peroxynitrite has been implicated in the activation of JNK in response to laminar shear stress in EC (25). Further investigation identified Akt-mediated eNOS phosphorylation as an important mediator of this pathway (24). Our finding that eNOS and c-Jun are phosphorylated in sheared FPAEC suggests that a similar pathway may be active in these cells. Because we were using the cloned human eNOS promoter in ovine PAEC, we confirmed the requirement for AP-1 in regulating the endogenous eNOS gene in FPAEC. Using the AP-1 antagonist curcumin, we were able to demonstrate, with RNase protection assays, that the shear-induced increase in eNOS mRNA expression was inhibited. This further strengthens our conclusion of the importance of the AP-1 cis-element in regulating shear-induced expression of eNOS in FPAECs.
Western blot analysis identified higher levels of eNOS in peripheral lung tissue from fetal lambs relative to adult sheep. Furthermore, eNOS levels were twofold higher in static FPAEC relative to static AdPAEC. Because equivalent levels of phosphorylated c-Jun were detected in static fetal and adult cells, it appears that AP-1 may not play a significant role in regulating basal eNOS transcription. Additional experiments are required to identify cis-elements within the eNOS promoter that are necessary for gene expression under static conditions. These studies may identify additional transcription factors that are developmentally regulated. NO levels were almost 20-fold higher in static FPAEC relative to static AdPAEC. These data suggest that eNOS is more active in the fetal cells, although the mechanisms that give rise to these basal differences are as yet unknown. eNOS enzyme activity is regulated by several proteins, including calmodulin and heat shock protein (HSP) 90 (21). The availability of substrate and cofactors may also account for differences in eNOS activity. Additional studies are therefore required to determine whether these molecules are also developmentally regulated in PAEC.
NO production was elevated after 1 h in both cell types with no corresponding change in eNOS protein, suggesting an increase in enzyme activity. We did not detect increases in phosphorylated Akt or phosphorylated eNOS after 1 h of shear, indicating that this pathway is not involved. The positive regulation of eNOS by calmodulin and HSP90 is enhanced by shear stress (21), raising the possibility that these proteins activate eNOS in the acute response to shear in PAEC. NO production increased eightfold in adult cells with only a 2.5-fold increase in fetal cells exposed to 1 h of shear. This suggests a greater increase in eNOS activity in the adult cells; since eNOS appears to be more active in resting fetal cells, the potential for further activation may be less. However, eNOS protein levels were twofold higher in fetal cells, but NO levels were about fivefold higher after 1 h of shear, indicating that eNOS enzyme activity is still higher in the fetal cells. The mechanisms that generate these developmental differences are unclear, although we have shown previously that NO production in FPAEC in response to acute shear stress involves PKC (59). Whether the same PKC pathway is involved in adult cells remains to be elucidated.
After 8 h of shear, eNOS and NO increased further, with the levels in sheared adult cells being similar to those in resting fetal cells. Akt-mediated phosphorylation of eNOS provides an additional means of increased NO production in fetal cells, although this pathway does not appear to be active in the adult cells. Surprisingly, NO levels were not significantly higher in fetal cells sheared for 8 h relative to cells sheared for only 1 h. This may be due to limited availability of the fluorophore at higher concentrations of NO and/or saturation of the images captured such that the software does not detect the true increase in fluorescence intensity. In a previous study we found a sevenfold increase and a 22-fold increase in NOx release in FPAEC sheared for 1 and 8 h, respectively (59). This suggests that we may be underestimating the amount of NO in FPAEC sheared for 8 h in this study.
We have demonstrated maturational changes in eNOS gene expression in ovine peripheral lung tissue, although the mechanisms involved are complex and poorly understood. Thus the objectives of this study were to identify developmental differences within PAEC and to characterize the shear stress response pathways in these cells. Factors that increase eNOS protein levels and enzyme activity are in effect in static fetal cells. Furthermore, by having nuclearly localized c-Jun, EC within the fetal pulmonary circulation appear to be primed to respond immediately to shear stress. Shear stress-induced activation of signaling pathways leading to the phosphorylation of c-Jun, Akt, and eNOS occurs only in FPAEC. These events are predicted to increase eNOS transcription and enzyme activity. This is likely to be of great importance considering that sufficient NO must be synthesized to decrease pulmonary vascular resistance and increase pulmonary blood flow at birth. By identifying the molecular events involved, we hope that better treatment strategies can be developed for conditions arising from an abnormal fetal-to-newborn transition.
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ACKNOWLEDGEMENTS |
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This research was supported in part by National Institutes of Health Grants HL-60190, HL-67841, and HD-398110; March of Dimes Grant FY00-98; and American Heart Association, Midwest Affiliates, Grant 0051409Z (all to S. M. Black). S. M. Black is a member of the Feinberg Cardiovascular Research Institute.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. M. Black, Northwestern Univ. Medical School, Ward 12-191, 303 E. Chicago Ave., Chicago, IL 60611-3008 (E-mail: steveblack{at}northwestern.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 January 17, 2003;10.1152/ajplung.00252.2002
Received 29 July 2002; accepted in final form 4 December 2002.
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