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J. Biol. Chem., Vol. 277, Issue 45, 42596-42602, November 8, 2002
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,From the Pulmonary and Critical Care Division, Department of Medicine/Tupper Research Institute, Tufts-New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02111 and the § Institute of Biochemistry, Medical School Hannover, 30625 Hannover, Germany
Received for publication, June 12, 2002, and in revised form, August 7, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Exposure to hypoxia causes structural changes in
the endothelial cell layer that alter its permeability and its
interaction with leukocytes and platelets. One of the well
characterized cytoskeletal changes in response to stress involves the
reorganization of the actin cytoskeleton and the formation of stress
fibers. This report describes cytoskeletal changes in pulmonary
microvascular endothelial cells in response to hypoxia and potential
mechanisms involved in this process. The hypoxia-induced actin
redistribution appears to be mediated by components downstream of MAPK
p38, which is activated in pulmonary endothelial cells in response to
hypoxia. Our results indicate that kinase MK2, which is a substrate of p38, becomes activated by hypoxia, leading to the phosphorylation of
one of its substrates, HSP27. Because HSP27 phosphorylation is known to
alter actin distribution in response to other stimuli, we postulate
that it also causes the actin redistribution observed in hypoxia. This
notion is supported by the observations that similar actin
redistribution occurs in cells overexpressing constitutively active MK2
or phosphomimicking HSP27 mutant. Overexpressing dominant negative MK2
blocks the effects of hypoxia on the actin cytoskeleton. Taken
together these results indicate that hypoxia stimulates the
p38-MK2-HSP27 pathway leading to significant alteration in the actin cytoskeleton.
Hypoxia causes injury in a variety of organs and has been
associated with many lung diseases including the acute respiratory distress syndrome, pulmonary embolism, and ischemia-reperfusion injury. Hypoxia has been shown to increase the permeability of the
endothelial barrier both in vitro (1-4) and in
vivo (5). Moreover, hypoxia increases endothelial adhesiveness to
neutrophils (6, 7). In that respect, endothelial cells respond to
hypoxia in a manner similar to their response to inflammation. However, as opposed to the response of endothelial cells to inflammatory products, which has been extensively explored, the signal transduction pathways involved in the endothelial response to hypoxia remain poorly
understood. Recent reports have demonstrated activation of the
stress-activated MAPK1 p38 in
response to hypoxia (8-16). For example, we have described the
activation of p38 in hypoxic pulmonary microvascular endothelial cells
and implicated it as one of the mechanisms of activation of the
reactive oxygen-producing enzyme, xanthine oxidase (16). The enzyme
MK2, immediately downstream of p38, is known to phosphorylate the small
heat shock protein HSP27 (17). Because HSP27 interacts with actin and
modulates cytoskeletal organization (18, 19), we investigated whether
the MK2 pathway is activated by hypoxia and whether this process can
lead to cytoskeletal changes. Our findings indicate that MK2 is indeed
activated by hypoxia in RPMEC, and that HSP27 phosphorylation is
increased concomitantly with reorganization of the actin cytoskeleton.
The effect of hypoxia on the actin cytoskeleton is mimicked by
overexpressing constitutively active MK2 and is blocked by
overexpressing dominant negative MK2 in endothelial cells. Furthermore,
overexpressing a phosphomimicking mutant HSP27 in endothelial cells
causes reorganization of the actin cytoskeleton similar to the actin
redistribution caused by hypoxia.
Cell Culture--
RPMEC were a gift from Dr. Una Ryan (Avant
Immunotherapeutics, Needham, MA) and have been well characterized by us
and others (20). These cells exhibit typical endothelial cobblestone
morphology and stain positively with antibodies against von Willebrand
factor. For hypoxic exposure, cells were placed in humidified airtight incubation chambers (Billups-Rothenberg, Del Mar, CA) and gassed with
3% O2, 5% CO2, balance N2.
Normoxic cells were placed in a tissue culture incubator maintained at
5% CO2 and 37 °C.
Actin Cytoskeleton Examination--
Cells were seeded on
poly-L-lysine- or collagen-coated coverslips. At various
degrees of confluence, cells in serum-free medium were subjected
to different treatments, e.g. hypoxia and/or kinase inhibitors. To control for the effect of coverslip coating, only cells
plated on the same substrate were compared and analyzed in a particular
experiment. At the end of the treatment, the coverslips were rinsed
twice with phosphate-buffered saline (PBS) and fixed for 10 min with
4% formaldehyde. Next, the coverslips were washed twice with PBS and
then permeabilized for 10 min with 0.4% Triton-X-100 in PBS. The cells
were stained with rhodamine-phalloidin (Molecular Probes, Eugene, OR)
for 20 min. The coverslips were then washed with PBS, mounted with
Citifluor, and examined using a Zeiss fluorescence microscope. The
amount of filamentous actin formed was assessed by quantifying
rhodamine-phalloidin fluorescence using image analysis software
from IP Lab Scanalytics (Fairfax, VA).
MK2 Kinase Assay--
After exposure of cells to normoxia or
hypoxia, activation of MK2 was assayed by measuring the activity of the
immunoprecipitated enzyme. Specific MK2 activity was assayed using the
MK2 assay kit from Upstate Biotechnology (Lake Placid, NY). In brief,
the cells were washed and lysed, and then MK2 was immunocomplexed with
agarose-conjugated anti-MK2 antibody by rocking overnight at 4 °C.
The immunocomplex was then brought down by centrifugation, and the
pellet was washed. Next, MK2-specific peptide substrate was added along
with [ Two-dimensional Electrophoresis and
Immunoblotting--
Isoelectrofocusing was performed in a Multiphor 2 unit according to manufacturer's instructions (Amersham
Biosciences). In brief, cells were lysed in 8 M
urea, 0.5% CHAPS, 60 mM dithiothreitol, 2% PharmalyteTM
4-7. Equal amounts of protein from cell lysates obtained from
different treatment groups were then mixed with IPGTM (Amersham
Biosciences) rehydration buffer and used to rehydrate ImmobilineTM
strips (pH 4-7 linear gradient; Amersham Biosciences). After
isoelectrofocusing, the strips were equilibrated with 2% SDS, 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v)
glycerol, 0.002% bromphenol blue, and 10 mg/ml dithiothreitol and then
with the same buffer containing 25 mg/ml iodoacetamide instead of
dithiothreitol. After equilibration, each strip was overlaid on a
single-well 10-20% gradient SDS-polyacrylamide gel and
electrophoresed according to Laemmli (21). After electrophoresis, the
gel was blotted onto an Immobilon-P membrane by electrophoretic
transfer. The membrane was then washed, blocked with 5% milk, and
probed with an antibody against HSP27 (Upstate Biotechnology). The
immunoreactive bands were visualized using anti-rabbit secondary
antibody conjugated to horseradish peroxidase and a chemiluminescent
substrate according to manufacturer's instructions (Super Signal, Pierce).
Transfection of Endothelial Cells--
Constitutively active
mutant and dominant negative mutant MK2 constructs as well as
phosphomimicking mutant HSP27 constructs were generated as described
(22-24). In brief, these constructs were made in the pcDNA3 vector
(Invitrogen), in which the cytomegalovirus promoter drives the
eukaryotic expression of the corresponding protein. The vectors used
for transfection were pcDNA3 vector alone, pcDNA3-MK2EE to
express constitutively active MK2 in which the residues T205E and T317E
were mutated, or pcDNA3-MK2KR to express dominant negative MK2 in
which the residue K76R was mutated. The vector pcDNA3-HSP27PM was
used to express HSP27 in which the residues S15D, S78D, S82D were
mutated to mimic phosphorylated HSP27. These vectors were introduced (5 µg) into endothelial cells by electroporation. Stable cell lines were
obtained by selection with geneticin, and resistant colonies were
isolated, expanded, and then screened for the level of MK2 activity or
for human HSP27 expression level.
Statistical Analysis--
Statistical analysis was carried out
using SPSS software (Chicago, IL). Student's t test was
used to analyze the differences between two groups. When comparisons
between multiple groups were carried out, one-way analysis of variance
was employed. Statistical significance was considered at
p < 0.05.
Hypoxia Alters the Actin Cytoskeleton in Pulmonary Endothelial
Cells--
Hypoxia is known to cause changes in the cytoskeleton that
alter the motility and permeability of the endothelial barrier. Using
rhodamine-conjugated phalloidin, which binds to filamentous actin, we
assessed the distribution of actin in normoxic and hypoxic cells. Our
results indicate that exposure of endothelial cells to hypoxia (3%
O2) causes a shift in filamentous actin from a web-like
structure (in normoxic cells) to parallel stress fibers. The latter
become thicker and increase in number with exposure time (Fig.
1A). The change is observed as
early as 30 min after exposure to hypoxia, becomes more significant by
1 h, and begins to reverse itself by 4 h of exposure to
hypoxia (Fig. 1A). In addition to the reorganization of
actin filaments, we observed an overall increase in filamentous actin
in response to hypoxia. To quantify filamentous actin,
rhodamine-phalloidin fluorescence was assessed in micrographs of
coverslips from several experiments using image analysis software as
described under "Experimental Procedures." As shown in Fig.
1B, there was a significant increase in filamentous actin by
1 h of hypoxia (150% as compared with normoxic control cells),
with a return to base line by 4 h of exposure. In conclusion,
these results indicate that hypoxia exerts a rapid and significant
change in the actin cytoskeleton.
Hypoxia Stimulates MK2 in Rat Pulmonary Microvascular Endothelial
Cells--
Recent reports have demonstrated the activation of the p38
MAPK in various cell types in response to hypoxia (8-16). Our
laboratory has recently demonstrated an important role for p38 in
mediating xanthine oxidase activation in response to hypoxia (16).
Because p38 kinase has been shown to phosphorylate and activate MK2,
and because MK2 is known to be involved in actin remodeling (17), we
examined the involvement of this latter kinase in mediating the effects
of hypoxia on the actin cytoskeleton. First, we tested whether MK2
becomes activated in hypoxic (3% O2) RPMEC. MK2 was immunoprecipitated from cell lysates with an agarose-conjugated antibody, and its activity was measured as described under
"Experimental Procedures." As shown in Fig.
2, MK2 activity increased in hypoxia with
a maximum increase observed after 1 h of hypoxia. The time course
of activation of MK2 was very similar to that of p38 activation by
hypoxia as observed in an earlier report (16). The increased MK2
activity was also blocked by pre-incubation with a p38 kinase inhibitor
(not shown). Hence, MK2, which is downstream of p38, appears to be
activated in response to p38 activation in hypoxia. The time course of
activation of MK2 is similar to that of actin cytoskeleton
reorganization in hypoxia (Fig. 1), consistent with a role for MK2 in
mediating that effect.
HSP27 Is Phosphorylated in Hypoxic Endothelial Cells--
Because
HSP27 is a known substrate of MK2, and has been shown to regulate the
actin cytoskeleton, we tested the possibility of HSP27 phosphorylation
in hypoxia. To check whether endogenous HSP27 phosphorylation is
increased in hypoxic RPMEC, cell lysates from normoxic and hypoxic (3%
O2) samples were analyzed by two-dimensional electrophoresis followed by immunoblotting with an antibody against HSP27. Phosphorylation causes a protein to become more acidic, thus
reducing its isoelectric point. Hence, differently phosphorylated HSP27
(non-, mono-, bi-, or triphosphorylated) can be resolved by
two-dimensional electrophoresis (25). Although phosphospecific antibodies have been used to study changes in HSP27 phosphorylation by
SDS-PAGE, these antibodies usually recognize one phosphoepitope and do
not discriminate between mono-, bi- or triphosphorylated HSP27. The
different spots shown in Fig. 3 reflect
putative nonphosphorylated as well as mono-, bi-, and triphosphorylated
forms of HSP27, with the triphospho-HSP27 being most acidic and
migrating farthest to the right. As shown in Fig. 3, by 30 min of
hypoxia, the relative amount of phospho-HSP27 increased significantly
compared with normoxia. By 1 h of hypoxia, there was a
significant decrease in the triphospho-HSP27 (Fig. 3). The distribution
of phospho- and nonphospho-HSP27 began to return to normal by 2 and
4 h of hypoxia (Fig. 3). The decrease in triphospho-HSP27 by
1 h was reproducible in different experiments, and its
significance is currently being investigated. Possibilities include
rapid aggregation or degradation of the most heavily phosphorylated
form. In conclusion, hypoxia caused a rapid increase in HSP27
phosphorylation by 30 min, which began to return to base line by 4 h of hypoxia. A comparison of the time course of actin redistribution
(Fig. 1) with the time course of HSP27 phosphorylation (Fig. 3) reveals
that stress fiber formation begins by 30 min of hypoxic exposure,
concomitant with HSP27 phosphorylation (Fig. 3). Stress fibers are
thickest by 1 h of hypoxia at a time when less HSP27 is available
to inhibit stress fiber formation. Finally, stress fibers become
thinner, and the actin resumes a normal distribution by 4 h of
hypoxia, which coincides with the reversal of HSP27 phosphorylation as indicated in Fig. 3.
MK2 Activity Is Correlated with Actin Cytoskeleton
Reorganization--
To assess the involvement of MK2 in mediating
hypoxia-stimulated alteration of the actin cytoskeleton, the level of
MK2 activity was modulated in endothelial cells. As no specific
inhibitors of MK2 are available, the activity of MK2 was modulated by
expressing different forms of the enzyme in cells. RPMEC were
transfected with the empty vector alone (mock-transfected), with
constitutively active MK2, or with dominant negative MK2. After
selecting stable transfectants with geneticin and isolating and
expanding clones, MK2 activity was measured as described under
"Experimental Procedures." As shown in Fig.
4, cells overexpressing the
constitutively active form of MK2 displayed significantly greater
activity compared with mock-transfected cells. Overexpressing dominant
negative MK2 mutant did not affect base-line MK2 activity (data not
shown).
Stably transfected cells overexpressing constitutively active MK2 and
dominant negative MK2 were plated on collagen-coated coverslips, and
then their actin cytoskeleton was examined as described above.
Overexpression of constitutively active MK2 caused an increase in
stress fiber formation (Fig.
5A) and filamentous actin
(Fig. 5B) in normoxic cells resembling the effect of
hypoxia. On the other hand, overexpressing dominant negative MK2
inhibited the formation of stress fibers in response to hypoxia (Fig.
6A). In the cells
overexpressing dominant negative MK2, no increase in filamentous actin
was observed in response to hypoxia (Fig. 6B). Thus, the
formation of actin stress fibers correlated with MK2 activity, and
disrupting MK2 activity blocked stress fiber formation in response to
hypoxia. These results are consistent with a role for MK2 in mediating
the effects of hypoxia on the endothelial cytoskeleton.
Overexpressing Phosphomimicking HSP27 Mutant Increases Stress Fiber
Formation--
To test whether phosphorylation of HSP27 has an effect
on actin distribution, RPMEC were transfected with the empty vector alone (mock-transfected) or with phosphomimicking HSP27 mutant in which
phosphorylatable amino acids were replaced by negatively charged
aspartates as described under "Experimental Procedures." After
selecting stable transfectants with geneticin and isolating and
expanding clones, the cells were grown on coverslips and labeled with
rhodamine-phalloidin as described above. Overexpression of the
phosphomimicking HSP27 mutant in endothelial cells caused an increase
in stress fibers and filamentous actin in normoxic cells (Fig. 5).
Thus, formation of stress fibers in endothelial cells correlates with
negatively charged amino acids, which mimic phosphorylated amino
acids in HSP27.
Hypoxia is associated with many lung diseases including acute
respiratory distress syndrome, pulmonary embolism, and
ischemia-reperfusion injury. The pulmonary microvascular endothelium is
an obvious target of hypoxia because of its key anatomical location in
the alveolar capillary gas exchange unit. In this report, we examined the role of downstream components of the p38 MAPK pathway in effecting structural changes in endothelial cells exposed to hypoxia. Our findings indicate that the kinase MK2 becomes activated in hypoxia leading to HSP27 phosphorylation. These changes are accompanied by
alterations in the filamentous actin cytoskeleton. A causal link
between MK2 activation, HSP27 phosphorylation, and actin redistribution
is supported by experiments in which the activity of MK2 and HSP27 were
modulated by overexpressing different forms of these proteins.
The endothelium constitutes a barrier that controls the flow of fluids
and materials from the blood to tissues, and it regulates blood vessel
tone, homeostasis, growth, and response to injury (for review, see Ref.
26). In response to injury, the structure and function of the
endothelium become altered in a manner that affects the physiology of
the blood vessel and the involved organ in general. For instance, the
permeability of the endothelial barrier has been shown to increase in
response to hypoxia, both in vitro (1-3), and in
vivo (5). Furthermore, hypoxia promotes the production of
cytokines and growth factors by the endothelial layer. For example,
interleukin-1 The mechanisms involved in vascular responses to hypoxia are likely to
be quite complex. Some of these responses involve the activation of
transcription through the action of transcription factors such as
hypoxia-induced factor-1 (HIF-1) (34-36). Other events, however, are
considered too rapid to be the result of transcriptional
processes. One example of a nontranscriptional hypoxic response by the
endothelium is the mobilization of P-selectin and its release from
membranous organelles, which allows it to bind and activate neutrophils
(7). Recent work from our laboratory identified another
nontranscriptional endothelial response to hypoxia, namely the
phosphorylation of the reactive oxygen-producing enzyme, xanthine
oxidase, and subsequent up-regulation of the enzymatic activity (16).
In these experiments, the rapid phosphorylation and activation of
xanthine oxidase was found to be mediated by the kinases casein kinase
II and p38 (16).
Activation of p38 MAPK has been described in various cell types in
response to hypoxia (8-15). Our laboratory has recently demonstrated
the activation of p38 by hypoxia in rat pulmonary microvascular
endothelial cells as well (16). p38 is a stress-activated MAPK that
becomes activated in response to different stimuli such as ultraviolet
radiation and hyperosmolarity. Because of its involvement in either
mediating the effects or regulating the expression of many growth
factors and cytokines that are important in inflammation, p38 has been
the subject of intensive research (for review, see Ref. 37). Inhibitors
of p38 have been developed for use in a wide variety of inflammatory
diseases from arthritis to lung disease (38). Once p38 is activated, it
can phosphorylate a variety of substrates, including the kinase MK2.
Upon its phosphorylation by p38, MK2 becomes activated and in turn
phosphorylates different substrates, such as the small heat shock
protein, HSP27. Expression of HSP27 is particularly high in the lung
(39, 40). Like other heat shock proteins, HSP27 can function as a
chaperone and is known to stabilize proteins such as citrate synthase
and alcohol dehydrogenase (41, 42). Of particular interest is the
ability of HSP27 to interact with actin and to reduce actin
polymerization into filaments (25). Upon phosphorylation, HSP27 changes
its polymer state such that it loses its function as a chaperone and no
longer blocks the polymerization of actin, thus resulting in the
stabilization of actin fibers (25). HSP27 has also been implicated in
the stabilization of actin fibers in vivo in ischemic rat
kidneys (43). Indeed, p38 has been implicated in mediating actin
reorganization through altering HSP27 phosphorylation in response to
oxidative stress and vascular endothelial growth factor (19, 44).
After demonstrating that p38 is activated in RPMEC by hypoxia (16), we
tested the hypothesis that downstream components of the p38 pathway
might mediate cytoskeletal change in hypoxia. First, we demonstrated
that activation of MK2 by hypoxia follows a time course that closely
mirrors the time course of p38 activation by hypoxia that we had
described previously (16). HSP27, a substrate of MK2, also becomes
phosphorylated within the same time frame. Furthermore, actin stress
fibers, which are known to be regulated by HSP27 phosphorylation,
become thicker and more abundant in response to hypoxia. These events
peak within 1 h of exposure to hypoxia. A causal link between
these events is supported by the results obtained with different MK2
and HSP27 constructs. Overexpressing constitutively active MK2 caused
actin redistribution similar to that observed in hypoxic cells.
Conversely, overexpressing dominant negative MK2 inhibited the
hypoxia-stimulated actin redistribution. These results support the
notion that hypoxia causes redistribution of the actin cytoskeleton
through activation of MK2. Furthermore, overexpressing a
phosphomimicking mutant HSP27 increased filamentous actin and stress
fiber formation, consistent with a direct role for HSP27
phosphorylation in mediating reorganization of the actin cytoskeleton.
Reorganization of the actin cytoskeleton has been associated with
changes in endothelial permeability and motility of endothelial cells,
as well as increased adhesiveness of inflammatory cells. Experiments
are currently under way to further elucidate how these processes might
be altered in our experimental system. In conclusion, MK2 and HSP27 are
important signaling molecules in mediating endothelial responses to
hypoxia such as cytoskeletal reorganization. The components of the
p38-MK2-HSP27 pathway may present targets for drug development against
diseases such as pulmonary edema, in which the endothelial barrier is altered.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and incubated with the
immunoprecipitated kinase with vigorous shaking at 30 °C. Then, the
complex was brought down by centrifugation, and the supernatant,
containing the peptide substrate, was spotted on p81 phosphocellulose
paper and washed with phosphoric acid and acetone to remove
unincorporated label. Finally, the p81 paper was transferred to
scintillation vials containing scintillation mixture, and the samples
were counted on a Packard beta-counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (56K):
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Fig. 1.
Hypoxia causes filamentous actin to switch
from a web-like distribution to parallel stress fibers. Stress
fiber formation was maximal at 1 h of hypoxia and returned to
normal by 4 h of hypoxia (A). There was also an overall
increase in filamentous actin, which was statistically significant
after 1 h of hypoxia (B). To assess the changes in
filamentous actin, rhodamine fluorescence was quantified in micrographs
from several experiments (n = 3-4 for each group)
using image analysis as described under "Experimental Procedures."
*, p < 0.05 versus mean of normoxic
control.
View larger version (12K):
[in a new window]
Fig. 2.
Hypoxia stimulates MK2 activity in
RPMEC. MK2 was immunoprecipitated, and its kinase activity was
assayed as described under "Experimental Procedures." Maximum
increase was observed at 1 h of exposure. *, p < 0.05 versus mean of normoxic control.
View larger version (18K):
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Fig. 3.
Hypoxia increases HSP27 phosphorylation by 30 min of exposure. Samples from different cell groups were analyzed
by two-dimensional electrophoresis as described under "Experimental
Procedures." Numbered positions refer to
putative phosphorylated forms: 1, nonphosphorylated;
2, monophosphorylated; 3, biphosphorylated;
4, triphosphorylated HSP27. The increase in phosphorylation
is followed by the disappearance of triphospho-HSP27 at 1 h of
hypoxia and a return to base-line expression at 2 and 4 h of
hypoxia.
View larger version (11K):
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Fig. 4.
Over-expressing MK2 in RPMEC. Cells
transfected with constitutively active MK2 had significantly higher MK2
activity compared with cells transfected with the empty vector. *,
p < 0.05.
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[in a new window]
Fig. 5.
Overexpressing constitutively active MK2 or
phosphomimicking mutant HSP27 causes a significant increase in stress
fibers in normoxic cells (A). Filamentous actin was
quantified by measuring rhodamine fluorescence in micrographs from
several experiments (n = 3-4 for each group) using
image analysis as described under "Experimental Procedures." There
was a statistically significant increase in filamentous actin in cells
overexpressing constitutively active MK2 and in cells overexpressing
phosphomimicking HSP27 as compared with mock-transfected cells
(B). *, p < 0.05 versus mean of
mock-transfected controls.
View larger version (61K):
[in a new window]
Fig. 6.
Overexpressing dominant negative MK2 blocks the
formation of stress fibers in response to hypoxia (A).
Filamentous actin was quantified by measuring rhodamine fluorescence in
micrographs from several experiments (n = 3-4 for each
group) using image analysis as described under "Experimental
Procedures." There was no statistically significant increase in
filamentous actin in response to hypoxia in cells overexpressing
dominant negative MK2 (B) as compared with mock-transfected
cell. *, p < 0.05 versus mean of
mock-transfected controls.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
production is increased in hypoxic endothelial cell
cultures (27). Vascular endothelial growth factor is a classic
hypoxia-induced angiogenic factor that mediates vascular remodeling in
the lung (28, 29). Hypoxia impairs endothelial anti-thrombogenic
potential (2) as well as the ability of the endothelium to regulate
vascular tone (26). For instance, the synthesis of the vasodilator
prostacyclin is decreased in hypoxic pulmonary artery rings as well as
in cultured endothelial cells from neonatal calves (30). Nitric oxide
(NO) is another endothelium-derived vasodilator regulated in hypoxia. Work from our laboratory has demonstrated regulation of the
constitutive as well as the inducible form of nitric-oxide synthase
(eNOS and iNOS, respectively) in response to hypoxia (31, 32). Hypoxia stabilizes iNOS mRNA expression induced by cytokine treatment (32),
suggesting that hypoxia may alter the effects of inflammatory cytokines
(33).
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FOOTNOTES |
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* This work was supported by grants from the American Lung Association, the Massachusetts Tobacco Control Program, and the National Institutes of Health (HL49441).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.
To whom correspondence should be addressed: Pulmonary & Critical Care Division, Tufts-New England Medical Center, 750 Washington St., No. 257, Boston, MA 02111. Tel.: 617-636-4352; Fax:
617-636-5953; E-mail: ukayyali@lifespan.org.
Published, JBC Papers in Press, August 28, 2002, DOI 10.1074/jbc.M205863200
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ABBREVIATIONS |
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The abbreviations used are: MAPK, mitogen-activated protein kinase; RPMEC, rat pulmonary artery microvascular endothelial cells; HSP27, heat shock protein 27; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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