From the Cardiovascular Pulmonary and Developmental
Lung Biology Research Labs and the
Department of Renal Medicine,
University of Colorado Health Sciences Center,
Denver, Colorado 80262
Received for publication, November 27, 2000, and in revised form, February 13, 2001
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ABSTRACT |
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Hypoxia has been shown to act as a
proliferative stimulus for adventitial fibroblasts of the pulmonary
artery. The signaling pathways involved in this growth response,
however, remain unclear. We tested the hypothesis that hypoxia-induced
proliferation of fibroblasts would be dependent on distinct (compared
with serum) activation and utilization patterns of mitogen-activated
protein (MAP) kinases initiated by G Change in the architecture of the blood vessel wall occurs in
response to a wide range of physiologic stimuli and various injurious
insults. In response to chronic hypoxic exposure, the pulmonary
arteries (PA)1 undergo
concentric thickening with medial and adventitial changes predominating
(1, 2). When the hypoxic exposure occurs in early neonatal life, the
adventitial changes are particularly striking and include early and
dramatic increases in fibroblast proliferation (1, 3, 4). Much of the
work done to date in defining this process has focused on the growth
factors that are produced under hypoxic conditions and their potential
effects on cell proliferation. Little work, however, has been done on the growth promoting signaling pathways that might be induced by
hypoxia itself and thus might affect vascular wall cell proliferation directly and/or markedly influence the response to locally produced growth factors. A better knowledge of this process is important for an
understanding of the remodeling process in many vascular diseases
because hypoxemic or ischemic conditions are thought to contribute to
this process.
Mitogen-activated protein (MAP) kinase family members including
extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase
(JNK), and p38 MAP kinase have been proposed to be important signaling
components linking extracellular stimuli to cellular responses
including cellular growth, differentiation, and metabolic regulation
(5-8). Mitogen-activated protein kinases can be activated by various
external stimuli such as growth factors, hormones acting via G
protein-coupled receptors, and physical stresses (6, 9). Hypoxia has
also been shown to stimulate ERK, JNK, and p38 MAP kinase in different
cell types (10-15). Recently, Jin et al. (16) demonstrated
the activation of all three MAP kinases in whole pulmonary arterial
wall of rat in response to hypoxia, although this study did not
evaluate the activation patterns in specific cell types in the vessel
wall. Activation of p38 MAP kinase has been demonstrated to be
necessary for the proliferation of adult bovine PA adventitial
fibroblasts and renal mesangial cells in response to hypoxia (12, 14).
By contrast, the mitogenic response to hypoxia of cultured human
osteoblastic periodontal ligament cells is mediated by the selective
phosphorylation and activation of ERK1/2 (15). These observations
support the hypothesis that the pathways used to achieve cell
proliferation in response to a specific stimulus may be cell
type-specific. There is insufficient information on the MAP kinase
pathways activated by hypoxia in cells at a developmental stage of
heightened susceptibility to hypoxia-induced change, i.e.
the neonate.
Moreover, little is known of the upstream signals that lead to MAP
kinase activation and ultimately cell proliferation in response to
hypoxia. One possible candidate is a family of heterotrimeric guanine
nucleotide binding proteins (G proteins). It has been shown that G
proteins can be activated by a number of environmental stimuli and play
critical roles in cell proliferation (17, 18). Previous work in our
laboratory demonstrated that specific subpopulations of smooth
muscle cells utilized pertussis toxin-sensitive G proteins (G We therefore hypothesized that hypoxia-induced proliferation of
neonatal PA adventitial fibroblasts is transduced through G
protein-mediated activation of MAP kinases in a manner that is
different from serum-induced MAP kinase activation and cell proliferation. To assess hypoxia-induced signaling, we utilized a
primary cell system generated from animals where hypoxia-induced fibroblast proliferation has been documented in vivo (1).
Cultured cells from these animals were amenable to serum deprivation
for 5 days, which allowed assessment of hypoxia-induced signaling in
the absence of exogenous stimuli that could influence the MAP kinase
signaling pathways. Further, an approach in which the effects of serum
and hypoxia were simultaneously evaluated in the same cells and was
utilized to determine whether there was a unique aspect of MAP kinase
signaling in hypoxia-induced proliferation.
Eagle's minimum essential medium (MEM), trypsin-EDTA 10×
suspension, penicillin, streptomycin, leupeptin, aprotinin,
phenylmethylsulfonyl fluoride, and Isolation and Growth of Adventitial Fibroblasts--
Adventitia
were harvested from the main pulmonary artery and aorta of 14-day-old
neonatal control calves. Fibroblasts were isolated, grown, and
characterized according to the previously described method (24). All
cells were maintained in MEM, pH 7.4, supplemented with 10% FBS, 100 units/ml penicillin, 0.1 mg/ml streptomycin and incubated in a
humidified atmosphere with 5% CO2 at 37 °C. Medium was
changed twice weekly, and cells were harvested with trypsin (0.2 g/liter) and EDTA (0.5 g/liter). Early passage (passages 1-6) cells
were used. The growth characteristics and light microscopic appearance
of the cells were unchanged up to passage 6.
Hypoxic Proliferative Response of Isolated Adventitial
Fibroblasts--
Fibroblasts were seeded at a density of 10 × 103 cells/cm2 in 10% FBS/MEM, allowed to
attach overnight and growth-arrested with MEM, 0.1% FBS for 5 days to
delete any possible proliferative effect of exogenous growth factors.
Cells were exposed to normoxic conditions (21% O2, 5%
CO2, balance nitrogen) or hypoxic conditions (either 10%
or 3-1% O2, 5% CO2, balance nitrogen)
at 37 °C in an airtight plexiglass chamber (Bellco Glass, Vineland,
NJ) in the presence of 1 µCi/ml [3H]thymidine for
24 h. DNA synthesis was measured as acid insoluble counts per
minute of [3H]thymidine (24) (Beckman LS 6500 scintillation counter; Ecoscint H mixture, National Diagnostics).
Results were expressed as cpm/cell × 103. For 72 h of hypoxic exposure, the chambers were purged and replenished daily
(20 min at 5 liters/min).
To test whether hypoxia affects the serum-stimulated growth response of
fibroblasts, cells were seeded sparsely (250 cells/cm2) in
10% FBS/MEM and allowed to attach overnight according to the
previously described method (25). From day 1, fibroblasts were exposed
to normoxia and hypoxia up to 11 days. The hypoxic chambers were
repurged with the gas mixtures every 24 h. Results were expressed
as cell counts × 103/well.
Phosphorylation of MAP Kinases in Response to Hypoxia--
Cells
(~500 × 103) were plated in 100-mm Petri dishes in
medium containing 10% FBS and growth-arrested for 5 days with 0.1% FBS/MEM. At the end of 5 days, medium was removed to leave 2 ml, so
that the cell monolayer was just covered, and then cells were exposed
to hypoxia (3% O2) for the indicated times (0, 10, 30, and
60 min and 24 h). Cells were lysed with the homogenization buffer
(20 mM Tris, pH 7.5, containing 0.25 M
sucrose, 3 mM EDTA, 3 mM EGTA, 50 mM mercaptoethanol, 50 µg/ml leupeptin, 50 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 0.1%
Triton X-100) (25), scraped off the dish, freeze-thawed, and
centrifuged at 6,000 rpm for 10 min at 4 °C. Supernatant was used as
whole cell lysates. Protein concentrations were determined by using a
Bio-Rad protein assay. 5-30 µg of protein were subjected to electrophoresis on 10% SDS-polyacrylamide gel and then transferred to
a polyvinylidene difluoride membrane. The membrane was blocked with 5%
milk in TBS-Tween for 1 h at room temperature and then incubated
with anti-phospho (active) MAP kinase antibodies in TBS-Tween with 5%
milk (1:500 to 1:1000) overnight at 4 °C. Antibodies against
activated MAP kinases were developed using their phosphorylation pattern at threonine/tyrosine residues. Anti-ERK1/2 is dually phosphorylated at Thr202/Tyr204, anti-JNK is
dually phosphorylated at Thr183/Tyr185, and
anti-p38 is dually phosphorylated at
Thr180/Tyr182. After three washes with
TBS-Tween, membranes were incubated with anti-rabbit antibodies
conjugated with horseradish peroxidase for 1 h at room
temperature. The bands were identified by using chemiluminescence
reagents, and the emitted light was recorded on film.
To compare the phosphorylation of MAP kinases in response to hypoxic
exposure and serum stimulation, the growth-arrested adventitial fibroblasts were stimulated with 10% FBS/MEM for similar lengths of
time as hypoxia. The cell lysates were analyzed for the phosphorylated and nonphosphorylated forms of MAP kinases by Western blot analysis. The protein loading for each sample was evaluated by stripping the
membrane with buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS,
100 mM 2-mercaptoethanol) and reprobed with anti-
Film images of Western blots obtained from ECL detection were analyzed
on a scanner and visualized using a Vista Scan software package.
Densitometric quantitation of the bands was performed using NIH Image
1.58 program. The bands were scanned, and the area under the curve of
each band was determined. This value represents the band intensity in
arbitrary units. The value at 0 h time was considered as 100%,
and fold increases in response to hypoxic exposure and serum
stimulation were calculated with respect to the 0 h.
Effects of JNK1 and JNK2 Antisense Oligonucleotides on Hypoxia
and Serum-induced Proliferative Responses of Fibroblasts--
All
oligonucleotides used in this study were phosphorothioate
oligonucleotides prepared according to a previously described method
(26). The transient transfection of fibroblasts with scrambled and
antisense constructs was performed using Lipofectin. Oligonucleotides
(200 nM) were mixed with Lipofectin (10 µl/ml) and added
to the cell monolayer. After 5-6 h, the medium was replaced with MEM
containing 0.1% FBS. For determination of JNK protein levels, cell
extracts were prepared with homogenizing buffer. An equal amount of
protein was resolved on SDS-10% polyacrylamide gels and visualized by
Western analysis with specific anti-JNK1 antibodies.
For the proliferation assay, growth-arrested fibroblasts were
transiently transfected with 200 nM of antisense and
scrambled oligonucleotides. As described above, after the 5 h of
lipofection, the medium was replaced with MEM supplemented with 0.1%
FBS. The next day, [3H]thymidine was added to the cells,
and cultures were either exposed to normoxia and hypoxia, or stimulated
with 10% FBS for 24 h. Cells were processed for the measurement
of DNA synthesis according to a previously described method (24).
Effects of PD98059 and SB202190 on Hypoxia-induced and
Serum-stimulated Growth Responses of PA Adventitial
Fibroblasts--
To detect the role of ERKs and p38 MAP kinases in
hypoxia-induced and serum-stimulated growth, fibroblasts were seeded at a density of 10 × 103/cm2 in 10%
FBS/MEM, grown for 1 day, and then growth-arrested for 5 days with
0.1% FBS/MEM. PD98059 (10 µM), specific blocker of ERK
(27), and SB202190 (100 nM), specific antagonist of p38 MAP
kinase (28), were added to the medium and incubated at 37 °C for
1 h. The cells were exposed to normoxia and hypoxia in the
presence of [3H]thymidine for 24 h and processed for
the measurement of DNA synthesis (24).
Effects of Pertussis Toxin on Proliferation and MAP Kinase
Activation of Fibroblasts in Response to Hypoxia--
To evaluate the
role of G protein in proliferative responses of fibroblasts, quiescent
cells were incubated with 100 ng/ml of pertussis toxin at 37 °C for
1 h and then either exposed to normoxia and hypoxia or stimulated
with 10% FBS for 24 h in the presence of
[3H]thymidine. DNA synthesis was measured according to
the previously described method (24).
To examine the function of G protein in hypoxia-induced activation of
MAP kinases, growth-arrested fibroblasts were incubated with 100 ng/ml
pertussis toxin overnight at 37 °C and then exposed to hypoxia.
Cells were harvested with homogenizing buffer for the preparation of
whole cellular lysates. 5-30 µg of protein were electrophoresed on
10% polyacrylamide gels followed by transfer to polyvinylidene
difluoride membrane and immunoblotting with anti-ERK, anti-JNK, and
anti-p38 MAP kinase antibodies detecting the phosphorylated forms.
Immunoreactive bands were visualized with ECL Western blotting
detection reagents.
Data Analysis--
All data are expressed as arithmetic
means ± S.E.; n equals the number of replicate
wells/test condition in representative experiments. One-way and two-way
analyses of variance followed by the Student-Newman-Keuls multiple
comparisons within and between groups of data points were utilized.
Data were considered significantly different if p < 0.05.
Hypoxia in the Absence of Exogenous Mitogens Induces Growth of
Adventitial Fibroblasts and Also Augments Serum-induced
Proliferation--
To evaluate the possibility that hypoxia itself
acts as a growth-promoting stimulus for fibroblasts isolated from the
PA adventitia of neonatal calves, we examined the effect of low oxygen
concentrations on DNA synthesis in fibroblasts in the absence of any
co-mitogens. Quiescent fibroblasts were exposed to gas mixtures with
varying amounts of oxygen (21%, 10%, and 3-1%) for 24 h
in the presence of [3H]thymidine, and DNA synthesis was
measured. We found that oxygen concentrations of 3-1%
initiated a 2-4-fold increase in thymidine incorporation in
fibroblasts compared with 21% O2 (Fig.
1A). To determine whether
hypoxia-induced increases in DNA synthesis resulted in a net increase
in cell number, growth-arrested cells were exposed to hypoxia (3%
O2 because this is where maximal DNA synthesis was
observed) for 72 h, and cell counts were performed. An increase in
the number of fibroblasts after chronic exposure to hypoxia was found
(Fig. 1B).
To determine whether the hypoxia-induced proliferative response was
unique to adventitial fibroblasts from the pulmonary circulation, we
compared the effects of hypoxia on DNA synthesis in aggregate fibroblast populations derived from both the PA and the thoracic aorta
of eight newborn calves. PA adventitial fibroblasts from all eight
calves consistently demonstrated an increase in replication upon
hypoxic exposure (Fig. 1C). In contrast, in six of eight animals, no increase in proliferation was observed in quiescent adventitial fibroblasts isolated from the aorta in response to hypoxia.
However, in two aortic fibroblast populations, hypoxia induced DNA
synthesis to the same levels as observed in PA adventitial fibroblasts
(Fig. 1C).
To evaluate whether hypoxia could effect the serum-induced
proliferative response of fibroblasts, growth curves in the presence and absence of hypoxia were evaluated. We found that the growth of
fibroblasts in serum was significantly enhanced under hypoxic conditions (Fig. 1D). These data suggest that
hypoxia-induced and serum-stimulated growth of fibroblasts might be
driven by distinct and additive/synergistic signaling pathways in
neonatal bovine PA adventitial fibroblasts.
Hypoxia Induces Activation of MAP Kinase Members in a Pattern
Distinct from That Induced by Serum--
In an effort to evaluate the
possibility that distinct signaling pathways are involved in
hypoxia-induced growth responses of fibroblasts, we examined the
effects of hypoxia on the activation of MAP kinase family members in
quiescent, serum-starved fibroblasts and compared them to those induced
by serum. Hypoxia induced a rapid and somewhat sustained activation of
both ERK1 and ERK2 (Fig. 2A).
The pattern of activation was similar for ERK1 and ERK2, although the
magnitude of change was greater for ERK1. Further, the time course of
activation for hypoxia was similar to that for serum, although the
magnitude of the activation was not as great as serum (Fig.
2B). At 24 h, hypoxia-exposed fibroblasts had slightly
higher levels of phospho-ERK1 and phospho-ERK2 than serum-stimulated
cells (Fig. 2). No modification in the protein level of total ERK
kinases was observed either in response to hypoxic exposure or serum
stimulation for up to 24 h (Fig. 2A), demonstrating
that the increase in ERK phosphorylation in response to both stimuli
was not due to an increase in total ERK protein.
JNK was transiently activated in fibroblasts following hypoxic
exposure (at 10 min) (Fig.
3A). p46 JNK appeared to
undergo a greater increase than p54 JNK. In serum-stimulated cells, JNK was also activated at 10 min, but the activation was maintained up to
30 min (Fig. 3A). Densitometric quantitation of the blots demonstrated that the increase in phosphorylation of p46 JNK in hypoxic
fibroblasts was ~8-fold greater than that of normoxic controls
(Fig. 3B). The increase in p46 JNK phosphorylation in response to serum reached its maximum after 10 min (15-fold) (Fig. 3B). The increases in phosphorylation of p54 JNK in response
to serum stimulation and hypoxic exposure were 7- and 5-fold,
respectively (Fig. 3C). Western blots using pan JNK antibody
demonstrated that JNK protein levels in adventitial fibroblasts did not
change during either hypoxia or serum exposure up to 24 h (Fig.
3A).
Using the same experimental paradigm, we also found that hypoxia
stimulated extensive phosphorylation of p38 MAP kinase (Fig. 4A). The hypoxia-induced
activation of p38 MAP kinase was biphasic. Activation was already
observed at 10 min, sustained up to 30 min, and then decreased to basal
levels. However, a second increase in phosphorylation of p38 MAP kinase
was observed in hypoxia-stimulated fibroblasts at 24 h. Serum
stimulated a rapid phosphorylation of p38 MAP kinase that was also
maintained up to 30 min (Fig. 4A). The maximum increases in
phosphorylation induced by hypoxia and 10% serum were 26- and 17-fold,
respectively (Fig. 4B). There was no effect of hypoxic
exposure on total p38 protein level for up to 24 h (Fig.
4A). The p38 protein levels were increased at 60 min and
24 h of serum stimulation in fibroblasts; however, at these time
points there was no detectable phosphorylation of the protein (Fig.
4B). Collectively these data demonstrate that both hypoxia
and serum activate ERK, JNK, and p38 MAP kinases, but there are
differences in the kinetics of activation of these kinases between the
two stimuli with the most striking differences being in p38 MAP kinase
activation.
MAP Kinase Activation Is Necessary for Hypoxia-induced
Proliferative Responses of PA Adventitial Fibroblasts--
The
hypoxia-induced increase in DNA synthesis in serum-starved fibroblasts
was completely attenuated by PD98059, a specific MEK inhibitor (26)
(Fig. 5A). To confirm that the
effects of PD98059 on DNA synthesis were direct and not caused by an
induction of apoptosis in quiescent fibroblasts, the rate of apoptosis
with and without PD98059 under both normoxic and hypoxic conditions was
evaluated by double staining of the cell nuclei with TUNEL and
Hoechst's dye. The apoptotic rate of growth-arrested fibroblasts was
not affected by PD98059 (data not shown), suggesting that ERK
activation is a necessary component of the hypoxia-induced growth
response of fibroblasts. In the same cells, serum-stimulated proliferation was only partially (20%) blocked by the same dose of
PD98059 (Fig. 5B), suggesting the existence of ERK
independent signaling pathways in the serum-stimulated growth response
of these fibroblasts.
Because pharmacological inhibitors of JNKs are currently not
commercially available, we employed specific antisense oligonucleotides designed to inhibit expression of specific JNK isoforms (JNK1 and JNK2)
and to determine whether activation of JNK was functionally related to
the hypoxia-induced proliferative response of fibroblasts (27). As
shown in Fig. 6A, the
antisense oligonucleotides of JNK1 inhibited the increase in
[3H]thymidine incorporation by fibroblasts in response to
hypoxia. In the presence of scrambled oligonucleotides, adventitial
fibroblasts maintained their unique hypoxia-induced proliferative
capability (Fig. 6A). We confirmed that antisense
oligonucleotides blocked the expression of JNK1 by demonstrating that
JNK1 protein levels were dramatically reduced in cells treated with the
antisense oligonucleotides (Fig. 6A). JNK1 antisense
oligonucleotides did not affect the rate of apoptosis under normoxic or
hypoxic conditions (data not shown). The role of JNK1 activation in
serum-stimulated growth responses of fibroblasts was also evaluated
using the antisense oligonucleotides. Serum-induced growth was also
attenuated by JNK1 antisense oligonucleotides (Fig. 6B). As
opposed to the inhibition of DNA synthesis observed with JNK1 antisense
oligonucleotides, JNK2 antisense oligonucleotides appeared to enhance
the hypoxia-induced proliferative responses of fibroblasts (Fig.
6C). In these experiments, the slight increase in apoptosis
observed under hypoxic conditions was blocked by JNK2 antisense
oligonucleotides (data not shown).
To evaluate the role of p38 MAP kinase in hypoxia-induced and
serum-stimulated proliferative responses of adventitial fibroblasts, we
used SB202190, a selective inhibitor of p38 MAP kinase (28). Pretreatment of cells with SB202190 significantly reduced the hypoxia-induced increase in DNA synthesis (Fig.
7A). To confirm that the
inhibitory effect of SB202190 was due to the inhibition of DNA
synthesis but not due to an increase in apoptosis, the apoptotic rate
of hypoxic fibroblasts were also examined in the presence and absence
of SB202190. The slight increase in apoptosis induced by hypoxia was
also blocked by SB202190, thus eliminating the possibility that the
inhibition of DNA synthesis was simply due to an increased rate of
apoptosis (data not shown). In contrast to the inhibition of
hypoxia-induced growth by SB202190, the serum-stimulated proliferative response of fibroblasts was potentiated by a similar concentration of the inhibitor (100 nM) (Fig.
7B), suggesting that activation of p38 MAP kinase has
distinct functional roles in hypoxia-induced and serum-stimulated
growth of fibroblasts.
We also evaluated hypoxia-induced activation patterns of ERK, JNK, and
p38 MAP kinase in aortic adventitial fibroblasts that lack the ability
to proliferate in response to hypoxia (see Fig. 1C,
nonresponsive cells). No increase in ERK and p38 MAP kinase activation
was observed in these cells in response to hypoxia. However,
hypoxia did induce a slight increase in JNK activation (1.5 fold) that
was of lesser magnitude than observed in cells induced to proliferate
by hypoxia (data not shown).
Pertussis Toxin-sensitive G Proteins Are Essential Upstream
Signaling Components of Proliferation and Activation of MAP Kinases in
Response to Hypoxia--
To evaluate the role of G
To determine whether the inhibitory effects of pertussis toxin on
hypoxia-induced proliferation were mediated through inhibition of MAP
kinases, quiescent fibroblasts were pretreated with pertussis toxin and
exposed to hypoxia, and MAP kinase activation was then evaluated using
antibodies against activated forms of the kinases. The hypoxia-induced
activation of ERK and JNK was almost completely blocked by pertussis
toxin (Fig. 8C). In contrast, pretreatment of fibroblasts
with 100 ng/ml pertussis toxin for 24 h had no effect on the
hypoxia-induced phosphorylation of p38 MAP kinase (Fig. 8C).
Therefore, hypoxia induces ERK and JNK through the activation of
G The present study demonstrates that hypoxia can
stimulate quiescent neonatal adventitial fibroblasts to proliferate in
the absence of any exogenous mitogens and that this proliferative response is dependent, in large part, on G Previous studies have demonstrated variable patterns of activation of
ERK, JNK, and p38 MAP kinase in response to hypoxia depending on the
cell type studied and the conditions under which the experiments were
done. Seko et al. (10, 11) demonstrated that hypoxia and
hypoxia/reoxygenation activate ERK as well as JNK and p38 MAP kinase in
cultured rat cardiac myocytes. However, the physiological end point of
hypoxia-induced activation of MAP kinases in these cells was not
examined. It has been reported that hypoxia activates JNK and p38 MAP
kinase and stimulates proliferation in adult bovine PA adventitial
fibroblasts (12, 29). Interestingly, in these cells hypoxia has little
effect on ERK. In PC12 cells, hypoxia has been shown to activate ERK
and p38 MAP kinase but not JNK (13). These observations suggest a
complex integration of signaling pathways in the regulation of cellular
responses upon hypoxic exposure that is cell type-specific and perhaps
even developmentally regulated. Our experimental design,
i.e. the withdrawal of serum for 5 days before hypoxic
exposure, was designed to allow assessment of MAP kinase signaling in
the absence of residual co-mitogenic stimuli. MAP kinase activity was
induced by hypoxia in cells that were also stimulated to proliferate.
In other cells, also from the adventitia of neonatal animals, hypoxia
did not induce proliferation, and MAP kinase activity was not
stimulated. Thus, the possibility exists that subsets of adventitial
fibroblasts exist that exhibit different sensitivities to MAP kinase
activation by hypoxia.
We have demonstrated a rapid and dramatic activation of ERK in
growth-arrested neonatal bovine fibroblasts that were stimulated to
proliferate with hypoxia, consistent with reports that these kinases
are critical for proliferation in a variety of cell types (30). A
relatively higher activation level of ERK1 compared with ERK2 was
observed in response to hypoxia, although under basal conditions, the
cells express a greater abundance of phosphorylated ERK2 than ERK1. A
high ratio of activated ERK1/ERK2 has been shown to be associated with
proliferation in muscle cells (31), supporting our hypothesis that
activation of ERK might be necessary for hypoxia-induced proliferation
in fibroblasts. The pattern of ERK activation was similar to that
observed under serum stimulation, although less in magnitude. In our
studies, inhibition of ERK activation during hypoxic exposure blocked
almost completely the hypoxia-induced proliferative responses of
fibroblasts. In contrast, serum-stimulated growth of fibroblasts was
only partially blocked by the inhibition of ERK activation. These
observations are consistent with the idea that ERKs play a more
critical role in hypoxia-induced proliferative response than in
serum-induced growth.
In our studies, activation of JNK occurred at 10 min of hypoxic
challenge. This is unlike observations in adult fibroblasts where
activation of JNK in response to hypoxia was not observed until after
3-6 h of hypoxic exposure (12). However, the time course of JNK
activation in response to hypoxia in our study is comparable with that
of the well known growth stimulus, serum. Three different JNK genes,
JNK-1, -2, and -3, and at least 10 different splice variants with
molecular weights between 46 and 54 exist. Our data suggest that
hypoxia, like serum, activates p46 JNK to a greater degree than p54
JNK. Many growth promoting factors, including serum, activate JNK (32).
However, most studies have suggested that this stress-activated pathway
is involved in growth inhibition rather than mitogenesis (33). Our
antisense oligonucleotide data suggest that JNK1 activation in response to both hypoxia and serum stimulation exerts positive effects on cell
proliferation, consistent with other reports demonstrating a role for
JNK1 in proliferation (27, 34, 35). In contrast, the JNK2 antisense
oligonucleotide results suggest that JNK2 might exert negative effects
on hypoxia-induced cell growth. Differential functional roles of JNK
isoforms have also been reported in cultured small cell lung cancer
(36). Thus, it is possible that hypoxia may actually simultaneously
activate positive and negative growth regulatory pathways as a
safeguard against excessive growth and that JNK1/2 are critical
components of this response.
We found a significant increase in p38 MAP kinase phosphorylation in
response to hypoxia. The biphasic activation pattern of p38 MAP kinase
was different than that observed for ERK and JNK and interestingly
different from the activation pattern induced by serum. The majority of
existing reports have suggested that p38 is involved in growth
inhibition (37, 38). However, a few reports have suggested positive
roles for this kinase in promoting cell hypertrophy (39), ischemic
preconditioning (40), and hemopoietic (41) and T cell proliferation or
differentiation (42, 43). Our results with the widely used pyridinyl
imidazole inhibitor of p38, SB202190, demonstrating attenuation of
hypoxia-induced proliferative responses of neonatal PA fibroblasts are
consistent with previous reports of the role of p38 MAP kinase in the
hypoxia-induced proliferative response of adult PA fibroblasts and
mesangial cells (14, 29). The fact that the same dose of SB202190 that
attenuated hypoxia-induced proliferation augmented serum-induced
proliferation also suggests potentially unique roles for p38 isozymes
in hypoxic cell responses. The p38 family of protein kinases consists
of several isoforms, including p38 Little is known of the upstream events necessary for MAP kinase
activation in response to hypoxia. In glioblastoma cell lines, hypoxia
induces activation of phosphatidylinositol 3-kinase/Akt pathway in a
PTEN-regulated manner (47). However, studies in carotid body and
PC12 cells have implicated hypoxia-induced activation of G proteins as
an early event in the modulation of ion channel activity and cell
depolarization (20-23). G In summary, we have shown that hypoxia stimulates proliferation of
certain adventitial fibroblast populations isolated from neonatal
calves in the absence of co-mitogens. Hypoxia-induced proliferation is
inhibited by pertussis toxin as is the activation of ERK and JNK,
implying an upstream role of Gi/o proteins.
We found that hypoxia stimulated increases in DNA synthesis and growth
of quiescent fibroblasts in the absence of exogenous mitogens and also
markedly augmented serum-stimulated growth responses. Hypoxia caused a transient activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the time course and
pattern of which was somewhat similar to that induced by serum but
which was of lesser magnitude. On the other hand, hypoxia-induced
activation of p38 MAP kinase was biphasic, whereas serum-stimulated
activation of p38 MAP kinase was transient, and the magnitude of
activation was greater for hypoxia compared with that of serum
stimulation. ERK1/2, JNK1, and p38 MAP kinase but not JNK2 were
necessary for hypoxia-induced proliferation because PD98059, SB202190,
and JNK1 antisense oligonucleotides nearly ablated the growth response. JNK2 appeared to act as a negative modulator of hypoxia-induced growth
because JNK2 antisense oligonucleotides led to an increase in DNA
synthesis. In serum-stimulated cells, antisense JNK1 oligonucleotides and PD98059 had inhibitory effects on proliferation, whereas SB202190 led to an increase in DNA synthesis. Pertussis toxin, which blocks G
i/o-mediated signaling, markedly attenuated
hypoxia-induced DNA synthesis and activation of ERK and JNK but not p38
MAP kinase. We conclude that hypoxia itself can act as a growth
promoting stimulus for subsets of bovine neonatal adventitial
fibroblasts largely through G
i/o-mediated activation of
a complex network of MAP kinases whose specific contributions to
hypoxia-induced proliferation differ from traditional serum-induced
growth signals.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
i/o) for growth to a far greater extent than other
smooth muscle cells populations and that these cell populations
proliferated in response to hypoxia, whereas the others did not (19).
Further, activation of G proteins has been speculated to be a critical early event in hypoxia-induced cell responses in different cell types
(20-23). However, the role of G proteins in hypoxia-induced activation
of MAP kinases and proliferative responses of vascular adventitial
fibroblasts is unknown.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol were purchased
from Sigma. Fetal bovine serum (FBS) was obtained from Gemini
Bio-Products, Inc. (Calabasas, CA). Molecular weight markers and
reagents for protein determination were from Bio-Rad. Antibodies
against phospho-ERK, phospho- and pan-JNK, phospho-p38, and PD98059
were obtained from New England Biolabs (Beverly, MA). Antibodies for
total p38, JNK1, and ERK1/ERK2 and horseradish peroxidase-conjugated
goat anti-rabbit and rabbit anti-goat immunoglobulin G were from Santa
Cruz Biotechnology (Santa Cruz, CA). Polyvinylidene difluoride membrane
was obtained from Amersham Pharmacia Biotech. Renaissance
chemiluminescence reagent plus was from PerkinElmer Life Sciences.
SB202190 was obtained from CalBiochem (San Diego, CA). Lipofectin was
from Life Technologies, Inc. [3H]Thymidine was from ICN
Biochemicals (Irvine, CA). Antisense and scrambled DNA constructs of
JNK1 (104492 and 104509, respectively) and JNK2 (101759 and 101760, respectively) were obtained from ISIS Pharmaceutical Company
(gift of Dr. Brett P. Monia). All the oligonucleotides are
phosphorothioate. The 5 nucleotides on both ends have
methoxyethyl modifications on the 2' position of the sugar, and the
middle 10 nucleotides are deoxy at the 2' position. Pertussis toxin was
from List Biological Labs (Campbell, CA).
-actin antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Hypoxia induces proliferation of neonatal
bovine adventitial fibroblasts in vitro.
A, hypoxic stimulation of DNA synthesis is greatest at
3-1% O2. Cells were plated at a density of 10 × 103/cm2 in 24-well plates and made quiescent by
serum deprivation (0.1% FBS) for 5 days. Cells were then exposed to
21, 10, and 3-1% O2 in the presence of
[3H]thymidine for 24 h. Values are the means ± S.E. for this and all subsequent figures. n = 4 replicate wells. *, p < 0.05 compared with 21%
O2 results. Similar results were obtained in three
independent experiments with cell populations isolated from at least
three independent animals. B, chronic hypoxic exposure
increases cell density above normoxic levels. Growth-arrested
fibroblasts were exposed to normoxia (21% O2) and hypoxia
(3% O2) for 3 days, trypsinized, and counted under
light microscope. n = 4 replicate wells. *,
p < 0.05 compared with cell counts under both normoxic
(24 and 72 h) and 24-h hypoxic exposure. C, hypoxia
consistently induces an increase in proliferation in all fibroblast
populations isolated from PA but only in selective fibroblast
populations derived from aorta. Adventitial fibroblasts were isolated
from both aorta and PA of the same neonatal calf and plated according
to the above-mentioned protocol. DNA synthesis in response to normoxia
(21% O2) and hypoxia (3% O2) was measured by
increase in [3H]thymidine incorporation. *,
p < 0.05 compared with corresponding normoxic value.
Results were obtained in independent experiments with cell populations
isolated from eight different animals. D, hypoxia augments
serum-stimulated growth of fibroblasts. Fibroblasts were plated at the
density of 250 cells/cm2 in medium containing 10% FBS and
allowed to attach overnight. On day 1, cells were exposed to normoxic
and hypoxic gas for 30 min/day for up to 11 days. *, p < 0.05 compared with the corresponding normoxic value. Similar results
were reproduced with at least two other cell populations.
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Fig. 2.
Hypoxia induces an increase in
phosphorylation of ERK1 and ERK2. A, Western blot
analysis of ERK1 and ERK2 phosphorylation in response to hypoxia and
10% serum. Representative immunoblots for phospho-ERKs and total ERKs.
Growth-arrested PA adventitial fibroblasts were stimulated with either
hypoxia (3% O2) or 10% FBS containing medium for 0, 10, 30, and 60 min and 24 h. Total protein was extracted and blotted
on polyvinylidene difluoride membrane. Blots were probed with an
anti-phospho p42/p44 antibody. Western blots were performed on the same
extracts using anti-ERK1 and -ERK2 antibodies to examine the level of
total amount of ERKs. Similar results were obtained from three separate
experiments. For each experiment, cells from different animals were
used. B, quantitative comparison of hypoxia and
serum-induced ERK1 phosphorylation in PA adventitial fibroblasts. The
bands on the radiographic film were scanned, and the area under the
curve was measured for individual band using NIH Image analysis
program. The value for 0 min of stimulation was considered as 100%.
C, quantitative comparison of hypoxia and serum-induced ERK2
phosphorylation in PA adventitial fibroblasts.
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Fig. 3.
Hypoxia stimulates transient activation of
JNK in PA adventitial fibroblasts. A, Western blot
analysis of phosphorylated and nonphosphorylated JNK in hypoxia and
serum-stimulated PA fibroblasts. Representative blots for phospho-JNK
and total JNK. Quiescent fibroblasts were exposed to either hypoxia or
10% FBS containing medium for 0, 10, 30, and 60 min and 24 h and
lysed with homogenizing buffer. Whole cell lysates were separated by
Western blot analysis and immunoblotted with anti-phospho and total JNK
antibodies. Similar results were obtained in three different
experiments. Cells used for the three experiments were isolated from
three different animals. B, quantitative comparison of
hypoxia and serum-induced p46 JNK phosphorylation in PA adventitial
fibroblasts. C, quantitative comparison of hypoxia and
serum-induced p54 JNK phosphorylation in PA adventitial
fibroblasts.
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Fig. 4.
Hypoxia induces biphasic activation of p38
MAP kinase in PA adventitial fibroblasts. A, Western
blot analysis of phosphorylated and total p38 MAP kinase in
hypoxia-induced and serum-stimulated fibroblasts. Representative blots
for phospho-p38 and total p38 MAP kinase. Growth-arrested PA
fibroblasts were stimulated with either hypoxia (3% O2) or
10% FBS for different lengths of time. Cell lysates were used for
immunoblotting with the antibodies against phospho and total p38 MAP
kinase. Similar results were obtained from three different experiments
using fibroblasts isolated from three different animals. B,
quantitative comparison of hypoxia and serum-stimulated p38 MAP kinase
phosphorylation in PA adventitial fibroblasts.
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Fig. 5.
Activation of ERK1/2 is necessary for hypoxic
proliferative responses in fibroblasts. A,
hypoxia-induced stimulation of DNA synthesis in fibroblasts is
abolished by inhibiting the activation of ERK1/2. Cells were seeded at
a density of 10 × 103/cm2 and
growth-arrested for 5 days. PD98059 (10 µM), a MEK
inhibitor that prevents the activation of ERK1 and ERK2, was added
1 h prior to hypoxic or normoxic exposure. DNA synthesis was
measured using [3H]thymidine. n = 4 replicate wells. *, p < 0.05 compared with normoxic
value; **, p < 0.05 compared with vehicle-treated
hypoxic results. Similar results were reproduced in at least two
independent cell populations. B, PD98059 only partially
inhibits serum-stimulated growth. Quiescent fibroblasts were pretreated
with the same dose of PD98059 (10 µM) as used in the
hypoxic cells and then stimulated with 10% FBS. Growth was assessed by
the measurement of DNA synthesis. n = 4 replicate
wells. *, p < 0.05 compared with unstimulated value;
**, p < 0.05 compared with vehicle-treated
serum-stimulated data. Similar results were obtained in two other
experiments using different cell populations.
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Fig. 6.
JNK1 and JNK2 have opposing effects in
hypoxia-exposed fibroblasts. A, JNK1 antisense
oligonucleotides markedly reduce JNK1 protein levels and inhibit
hypoxia-induced proliferative responses of fibroblasts.
Inset, total JNK1 protein in control fibroblasts
(C), in fibroblasts treated with scrambled (SCR)
oligonucleotides, and fibroblasts treated with antisense JNK1
oligonucleotides (AS) and the cell lysates immunoblotted
with anti JNK1 antibody. Cells were plated at a density of 25 × 103/cm2 and growth-arrested for 5 days. On day
4, cells were transfected with JNK1 (scrambled and antisense
oligonucleotides, 200 nM) and Lipofectin for 6 h. On
day 5, cells were exposed to normoxia (21% O2) and hypoxia
(3% O2) for 24 h in the presence of
[3H]thymidine. B, serum-stimulated
proliferative responses of fibroblasts are inhibited by JNK1 antisense
oligonucleotides. Growth-arrested cells were transiently transfected
with either JNK1 (scrambled and antisense) oligonucleotides as above,
stimulated with 10% FBS and DNA synthesis was measured. C,
JNK2 antisense oligonucleotides augment hypoxia-induced proliferative
responses of fibroblasts. Quiescent fibroblasts were transfected with
JNK2 (scrambled and antisense, 200 nM) oligonucleotides,
exposed to hypoxia and [3H]thymidine incorporation was
examined. n = 4 replicate wells. *, p < 0.05 compared with normoxic results in the presence of scrambled
oligonucleotides; **, p < 0.05 compared with hypoxic
value in the presence of scrambled oligonucleotides. Similar results
were reproduced in at least two different cell populations for all data
in the figure.
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Fig. 7.
P38 MAP kinase play a role in hypoxia-induced
proliferation. A, SB202190, an inhibitor of p38 MAP
kinase, attenuates hypoxia-induced augmented growth responses of
fibroblasts. Cells were plated at a density of 10 × 103/cm2 and growth-arrested for 5 days.
SB202190 (100 nM) was added, incubated at 37 °C for 30 min, and exposed to normoxia (21% O2) and hypoxia (3%
O2) in the presence of [3H]thymidine for
24 h. B, serum-stimulated growth of fibroblasts is
augmented by SB202190. Quiescent fibroblasts were pretreated with the
same concentration of SB202190 (100 nM) as used in hypoxia
and then stimulated with 10% FBS, and proliferation was examined by
measuring DNA synthesis. n = 4 replicate wells. *,
p < 0.05 compared with normoxic results in the
presence of vehicle; **, p < 0.05 compared with
hypoxic results in the presence of vehicle. Similar results were
reproduced in a minimum of two separate cell populations.
i/o
protein in hypoxia-induced proliferative responses, PA adventitial
fibroblasts were treated with pertussis toxin, which inhibits
Gi/Go activation by ADP-ribosylation. The
hypoxia-induced increase in DNA synthesis was markedly attenuated by
pertussis toxin (Fig. 8A).
Proliferation in response to serum stimulation was also attenuated by
pertussis toxin (Fig. 8B), demonstrating an important role
for G
i/o activation in both hypoxia-induced and
serum-stimulated growth responses of neonatal adventitial fibroblasts.
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Fig. 8.
Inhibition of
G i/o proteins by pretreatment with
pertussis toxin attenuates hypoxia-induced stimulation of DNA synthesis
and selectively inhibits MAP kinase activation. A,
hypoxia-induced increase in [3H]thymidine incorporation
in PA fibroblasts is abolished in the presence of pertussis toxin.
Pertussis toxin (100 ng/ml) was added to growth-arrested adventitial
fibroblasts and incubated for 1 h, and then the cells were exposed
to either normoxia or hypoxia in the presence of
[3H]thymidine for 24 h. B,
serum-stimulated growth of fibroblasts is attenuated by pertussis
toxin. Quiescent fibroblasts were pretreated with pertussis toxin (100 ng/ml, 1 h) and stimulated with 10% FBS, and growth was measured
by DNA synthesis. n = 4 replicate wells. *,
p < 0.05 compared with normoxic results; **,
p < 0.05 compared with hypoxic results. Similar
results were reproduced in three independent experiments using cell
populations isolated from separate animals. C,
hypoxia-induced activation of ERK and JNK but not p38 MAP kinase is
blocked by pertussis toxin. Representative blots for phospho-ERK,
phospho-JNK, and phospho-p38 MAP kinase. Growth-arrested fibroblasts
were treated with 100 ng/ml pertussis toxin overnight and then exposed
to hypoxia. Cell lysates were separated by Western blot analysis and
probed with the antibodies against phospho-ERK, phospho-JNK, and
phospho-p38 MAP kinase. Similar results were reproduced in a minimum of
two other fibroblast populations.
i/o proteins, whereas hypoxia-induced activation of p38
MAP kinase occurs through other G proteins or other as yet undefined pathways.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
i/o-mediated
activation of ERK and JNK pathways and G
i/o-independent
activation of p38 MAP kinase. Equally as important is the observation
that not all adventitial fibroblast populations posses the capability
of proliferating in response to hypoxia and that in the unresponsive
cells hypoxia does not stimulate ERK or p38 activation. We showed
that the hypoxia-induced proliferative response appears to be mediated
through different mechanisms than those activated when the cells are
stimulated to proliferate with serum. This was demonstrated by
experiments showing that: 1) activation patterns of ERK, JNK and
especially p38 MAP kinases were different in response to hypoxia
versus serum; 2) blocking G
i/o, ERK, and JNK1
nearly ablated the proliferative response induced by hypoxia yet had
only moderate effects on serum-induced proliferation; 3) blocking p38
MAP kinase with SB202190 attenuated hypoxia-induced proliferation
yet led to an increase in serum-induced growth; and 4) hypoxia led to
synergistic augmentation of serum-induced growth.
, p38
, p38
2, p38
, and
p38
(44). The inhibitor, SB202190, is nearly equipotent against p38
and p38
but does not inhibit p38
or p38
(45).
Therefore, the inhibitory effects of SB202190 on the hypoxia-induced
growth response of fibroblasts suggest that activation of selective p38 isoforms might play an important role in hypoxic growth responses of
fibroblasts. Selective activation of p38
and p38
isoforms in
response to hypoxia has also been demonstrated in PC12 cells (46).
Future studies will be aimed at delineating the specific activation
patterns of p38 MAP kinase isoforms and their role in cellular
responses upon hypoxic exposure in neonatal adventitial fibroblasts.
i proteins have been shown to
be involved in mediating mitogenic responses and to be activated by
environmental stresses such as shear stress, mechanical stretch, and
reactive oxygen species (16, 17, 48). Previous experiments have also
established that certain subtypes of vascular wall cells utilize
G
i/o proteins to a far greater degree than others for
proliferative responses (18). Our results with pertussis toxin, which
inhibits signaling through G
i/o, strongly supports the
hypothesis that activation of G
i/o proteins is necessary for hypoxia-induced proliferation. Our data also demonstrate that hypoxic activation of ERK and JNK is mediated through
G
i/o, but the activation of p38 MAP kinase occurs
independent of this protein. It is possible that hypoxia might also
activate pertussis toxin-insensitive G proteins, e.g.
G
q/11 or G
12/13, and hypoxia-induced
activation of p38 MAP kinase might be mediated through these G
proteins. Heterogeneous involvement of G proteins in hypoxia-induced
activation of MAP kinases is consistent with the observation that
activation of ERK but not p38 MAP kinase appears to be dependent at
least in part on G
i proteins during contraction of
collagen matrices by fibroblasts under isometric tension (49).
Stimulation of the sphingosine 1-phosphate receptor has also recently
been shown to be coupled to activation of ERK and p38 by pertussis
toxin-sensitive and -insensitive mechanisms, respectively (50).
Pertussis toxin-sensitive G proteins have also been implicated in
activation of ERK in cardiac and endothelial cells subjected to changes
in mechanical forces (51, 52). Thus, our results indicate that
G
i/o proteins are a necessary upstream component of
hypoxia-induced proliferation in neonatal fibroblasts. Further, our
observations that hypoxia in the absence of exogenous ligands
stimulates MAP kinase activation and cell proliferation raises the
possibility that G protein activation and subsequent cellular events
occur in a ligand-independent manner. This is consistent with recent
observations of Nishida et al. (48), who found that the
G
-responsive ERK activation induced by
H2O2 is independent of ligands binding to
Gi-coupled receptors. Future work will be directed at
evaluating the pathways through which hypoxia activates G proteins.
i/o proteins in hypoxic
growth regulation. Importantly, the MAP kinase signaling pathways
induced by hypoxia, especially p38 MAP kinase, appear distinct from
that induced by serum, allowing potentially additive or synergistic
interactions with growth factors under pathologic conditions. Further,
our results suggest the existence of different subpopulations of
fibroblasts, only some of which are capable of transducing a hypoxic
stimulus into a proliferative response.
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ACKNOWLEDGEMENTS |
---|
We thank Steve Hofmeister and Sandi Walchak for harvesting bovine pulmonary artery tissue, Marcia McGowan for final manuscript preparation, Dr. Michael Zawada for critical review of this manuscript, and Isis Pharmaceuticals for providing the JNK1/JNK2 antisense oligonucleotides.
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FOOTNOTES |
---|
* This work was supported in part by Specialized Center of Research Grant HL 56481 and National Institutes of Health Grant HL 14985.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.
§ Supported by a postdoctoral fellowship from the American Heart Association, Arizona, Colorado and Wyoming Affiliate, a Giles Filley Research Award from the American Physiological Society, and a research grant from the American Lung Association. To whom correspondence should be addressed: Campus Box B-131, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262. E-mail: Mita.Das@uchsc.edu.
¶ Supported by National Institutes of Health Training Grant HL 07171.
** Supported by National Institutes of Health Grants DK 39902, DK 19928, and HL 62924.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M010690200
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ABBREVIATIONS |
---|
The abbreviations used are: PA, pulmonary artery; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MEM, Eagle's minimum essential medium; FBS, fetal bovine serum.
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