From the Departments of b Surgery, and c Physiology and Cellular Biophysics, d Herbert Irving Comprehensive Cancer Center, and the Departments of h Pharmacology and j Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032, the e Division of Nephrology, Department of Medicine, Oregon Health Sciences University, Portland, Oregon 97201, the f Section of Cardiology, Department of Medicine, University of Illinois at Chicago, Chicago Illinois 60612, the g James A. Haley Veterans Hospital, University of South Florida, Tampa, Florida 33711, and the i Departments of Immunology and Vascular Biology, Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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The paradigm for the response to
hypoxia is erythropoietin gene expression; activation of
hypoxia-inducible factor-1 (HIF-1) results in erythropoietin
production. Previously, we found that oxygen deprivation induced tissue
factor, especially in mononuclear phagocytes, by an early growth
response (Egr-1)-dependent pathway without
involvement of HIF-1 (Yan, S.-F., Zou, Y.-S., Gao, Y., Zhai, C.,
Mackman, N., Lee, S., Milbrandt, J., Pinsky, D., Kisiel, W., and Stern,
D. (1998) Proc. Natl. Acad. Sci.
U. S. A. 95, 8298-8303). Now, we show
that cultured monocytes subjected to hypoxia
(pO2 The cellular response to oxygen deprivation involves a series of
metabolic and biosynthetic events associated with adaptation to a
hypoxic environment. Enhanced expression of the
noninsulin-dependent glucose transporter GLUT1, key
glycolytic enzymes, erythropoietin, and vascular endothelial growth
factor are well described examples of the host response to hypoxia with
obvious advantages for survival in an oxygen-deficient environment (1).
Each of these events appears, in large part, to be mediated by the
transcription factor hypoxia-inducible factor-1
(HIF-1)1 (1), which is also
implicated in angiogenesis, based on the phenotype of
HIF-1 Venous thrombosis is associated with local hypoxemia and stasis, and
has a considerable morbidity and mortality. Although cessation of blood
flow perturbs a spectrum of metabolic and hemodynamic factors, we have
found that normobaric hypoxia causes de novo expression of
tissue factor, especially in mononuclear phagocytes (MPs), resulting in
fibrin deposition in pulmonary vasculature (17, 18). Furthermore, we
have observed recently that hypoxia-mediated expression of tissue
factor is caused by the early growth response gene product Egr-1, based
on the parallel absence of enhanced tissue factor expression and fibrin
deposition in the pulmonary vasculature of homozygous Egr-1
null mice (19). These observations suggested the possible contribution
of Egr-1 to the cell biology of hypoxia, potentially
engaging a pathway with quite different outcomes for the adaptive host
response than HIF-1. In this study, we analyzed up-regulation of
Egr-1 in hypoxic MPs and have found it to occur at the
transcriptional level, consequent to activation of protein kinase
C- Cell Culture and Induction of Hypoxia--
A line of rat
mononuclear phagocytes (NR8383; alveolar macrophages) was obtained from
ATCC (Manassas, VA), and cells were grown in Ham's F-12K medium
containing 15% heat-inactivated fetal calf serum. Human blood
monocytes were harvested from peripheral blood by density gradient
centrifugation and plated for 2 days in RPMI 1640 medium containing
10% fetal calf serum (11). The human monocyte line U937 (ATCC) was
also grown in RPMI 1640 with fetal calf serum (10%). Mouse macrophages
(ATCC; P388D1) were grown in high glucose RPMI 1640 containing 10%
fetal calf serum. Wild-type hepatoma cells (Hepa1c1c7) or hepatoma
cells deficient in the ARNT/HIF-1
Experiments employing mice subjected to hypoxia (final oxygen
concentration of 5.5-6.5%) employed C57BL6/J mice (12-15 weeks old;
Jackson Laboratories, Bar Harbor ME) and utilized a specially built
environmental chamber (17).
Analysis of Egr-1 Expression in Hypoxic Murine
Lung--
Following hypoxia, mice were killed, and tissue was
processed immediately. For Northern analysis, tissue was cut into small pieces, immersed in Trizol (Life Technologies, Inc.), homogenized, and
total RNA was extracted and subjected to electrophoresis (0.8% agarose). RNA was transferred to Duralon-UV membranes (Stratagene), and
membranes were then hybridized with 32P-labeled cDNA
probe for mouse Egr-1 (21). Blots were also hybridized with
32P-labeled
For Western blotting, nuclear extracts were prepared (see below) and
subjected to SDS-PAGE (7.5%). Proteins in the gel were transferred
electrophoretically to nitrocellulose membranes, and immunoblotting was
performed with rabbit anti-Egr-1 IgG (Santa Cruz Biotechnology, Santa
Cruz, CA) and anti-Sp1 IgG (Santa Cruz Biotechnology) according to the
blotto procedure (23). Sites of primary antibody binding were
visualized with horseradish peroxidase-conjugated goat anti-rabbit IgG
(Amersham International, Buckinghamshire, U. K.). The final detection
of immunoreactive bands was performed using the enhanced
chemiluminescent Western blotting system (Amersham).
For immunocytochemical studies, lung tissue was harvested, cut into
small pieces, washed with phosphate-buffered saline (pH 7.0) to remove
blood, fixed in formalin, and embedded in paraffin (19). Sections were
first stained with primary antibodies, anti-Egr-1 IgG (Santa Cruz
Biotechnology), rat F4/80 monoclonal antibody (Caltag Laboratories,
South San Francisco, CA), or rabbit anti-smooth muscle
The electrophoretic mobility gel shift assay (EMSA) was performed on
nuclear extracts prepared immediately from hypoxic lung using the
method of Dignam et al. (24). Double-stranded
oligonucleotide probes for Egr (Santa Cruz Biotechnology)
were 5'-end labeled with [32P]ATP by using T4
polynucleotide kinase and standard procedures. Binding reactions were
performed as described (9), and samples (5 µg of protein in each
lane) were loaded directly onto nondenaturing polyacrylamide/bisacrylamide (6%) gels. Gels were prerun for 20 min
before samples were loaded, and electrophoresis was performed at room
temperature for 1.5-2 h at 100 volts. For competition studies, a
100-fold molar excess of unlabeled probes for NF-IL-6 (9), HIF-1 (1),
Egr (Santa Cruz Biotechnology), or AP-1 (Santa Cruz
Biotechnology) was added.
Analysis of Egr-1 Expression in Cultured MPs--
MPs were
subjected to hypoxia for the indicated times, and total RNA was
extracted using Trizol and subjected to Northern analysis. The same
protocol as for Egr-1 analysis in mouse lung was employed
(see above). Where indicated, Northern analysis was also performed to
detect tissue factor transcripts. In this case, 32P-labeled
tissue factor cDNA was employed to probe membranes with immobilized
RNA. Nuclear run-on analysis, to assess the rate of Egr-1
transcription, was performed on cultured MPs deprived of serum for
24 h and then subjected to normoxia or hypoxia for 30 min. Nuclei
were isolated, and in vitro transcription was performed as
described (9). EMSA using the consensus 32P-labeled
Egr probe on nuclear extracts from hypoxic cultured MPs
employed the same methods as for lung tissue above. Transient transfection, with Superfect Reagent (Qiagen, Chatsworth, CA), used a
series of deletional/truncated Egr-1 promoter-reporter (luciferase) constructs: A (the designation given in Fig.
4A) includes a 1.2-kilobase fragment of the murine
Egr-1 5'-flanking sequence Egr-1(1.2)Luc
(25-26); B includes sequences from 1 through 386 and from 886 through
1000; C includes sequences from 511 through 1000; D includes sequences
from 511 through 686 and 886 through 1000; E includes sequences from
811 through 1000; F includes sequences from 845 through 1000; and G
includes sequences from 866 through 1000. The construct labeled
V in Fig. 4A denotes the promoterless vector pXP2
(23). The constructs pYSF62&65 were comprised of Analysis of the Role of PKC
Transfection assays employed promoter-reporter constructs to monitor
transcription of Egr-1 (pYSF62, see above) and tissue factor
(designated as pTF[
Subcellular association of PKC Delineation of the Pathway Leading to Expression of Egr-1 in
Cultured MPs Subjected to Hypoxia--
Cotransfection of MPs used the
same procedure as above. To analyze the role of raf, cells were
transfected with a dominant-negative (DN)-Raf (pCGN raf N4;
generously provided by Dr. Channing Der, University of North Carolina,
Chapel Hill) (36). The MEK inhibitor PD98059 (New England Biolabs) was
employed at a concentration of 10 µM and was added 1 h before placement of cells in hypoxia. Other constructs included
DN-ERK 1 (pCMV-ERK 1[K>R]), DN-ERK 2 (pCMV-ERK 2[K >R]), and
DN-Elk-1 (pCMV-Elk-383A) (all generously provided by Dr. Peter Shaw,
Queen's Medical Center, Nottingham, UK) (38). The Elk-1 chimeric
reporter system was employed to detect activation of Elk-1 (39); the
GAL4-Elk-1 expression vector and the 5xGAL4-E1b-luciferase reporter
were generously provided by Dr. Richard Maurer (Oregon Health Sciences
University, Portland). In-gel kinase assays were used to monitor
activation of for ERK 1/2 (40). In brief, U937 cells in serum-free
medium were exposed to hypoxia for 10 min, and cells were washed three
times in ice-cold phosphate-buffered saline and suspended in ice-cold
extraction buffer. Cell extracts were resolved on SDS-PAGE (10%)
containing 0.5 mg/ml myelin basic protein (Sigma).
Data Analysis--
Significant differences between experimental
groups were detected using analysis of variance for unpaired variables,
with post hoc comparisons performed using Tukey's
procedure. Data that are shown in graphical format represent the
means ± S.E., with a p < 0.05 between groups
considered statistically significant.
Expression of Egr-1 in Lungs of Mice Subjected to
Hypoxia
Mice exposed to hypoxia displayed time-dependent
induction of Egr-1 mRNA evident by 15 min, with maximal
expression by 30 min (Fig.
1A). Western analysis of
nuclear extracts from hypoxic lung showed an increase in Egr-1 antigen,
with a major immunoreactive band corresponding to a molecular mass of
12 torr) displayed increased
Egr-1 expression because of de novo
biosynthesis, with a
10-fold increased rate of transcription. Transfection of monocytes with Egr-1 promoter-luciferase
constructs localized elements responsible for hypoxia-enhanced
expression to
424/
65, a region including EBS (ets binding site)-SRE
(serum response element)-EBS and SRE-EBS-SRE sites. Further studies
with each of these regions ligated to the basal thymidine kinase
promoter and luciferase demonstrated that EBS sites in the element
spanning
424/
375 were critical for hypoxia-enhanceable gene
expression. These data suggested that an activated ets factor, such as
Elk-1, in complex with serum response factor, was the likely proximal trigger of Egr-1 transcription. Indeed, hypoxia induced
activation of Elk-1, and suppression of Elk-1 blocked up-regulation of
Egr-1 transcription. The signaling cascade preceding Elk-1
activation in response to oxygen deprivation was traced to activation
of protein kinase C-
II, Raf, mitogen-activated protein
kinase/extracellular signal-regulated protein kinase kinase and
mitogen-activated protein kinases. Comparable hypoxia-mediated
Egr-1 induction and activation were observed in cultured
hepatoma-derived cells deficient in HIF-1
and wild-type hepatoma
cells, indicating that the HIF-1 and Egr-1 pathways are
initiated independently in response to oxygen deprivation. We propose
that activation of Egr-1 in response to hypoxia induces a
different facet of the adaptive response than HIF-1, one component of
which causes expression of tissue factor, resulting in fibrin deposition.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/arylhydrocarbon receptor nuclear translocator (ARNT) and
HIF-1
deletionally mutant mice (2-4). Although HIF-1 is critical
for restoring cellular homeostasis in hypoxia, biosynthetic events
triggered by oxygen deprivation extend beyond HIF-1. For example,
hypoxia leads to the induction of c-Fos and appearance of c-Jun/c-Fos
AP-1 heterodimers in cultured cells, potentially promoting expression
of a range of genes, especially those involved in cell growth (5-8).
Oxygen deprivation also activates the transcription factor C/EBP
(9,
10), which, on binding to nuclear factor-interleukin 6 (NF-IL-6)
motifs, results in expression of IL-6, a cytokine associated with acute
inflammation. Finally, some investigators have observed activation of
NF-
B in hypoxia (11-15), although others have found such activation
only during the immediate reoxygenation period associated with oxidant
stress (16). These considerations underscore the complexity of the
cellular response to environmental challenge by hypoxic and/or
oxidative stress.
II (PKC
II), leading to Raf- and MEK-dependent activation of MAP kinases, ERK 1/2, and, ultimately, activation of the
ets factor Elk-1. Although the physiologic and pathophysiologic consequences of Egr-1 activation in hypoxia are not yet
clear, induction of the trigger of the procoagulant pathway, tissue
factor, suggests the basis for pathologic events underlying thrombotic pathology associated with oxygen deprivation by a pathway distinct from
HIF-1.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
function (c4) were generously
provided by Dr. Oliver Hankinson (UCLA) and were grown in
-minimal
essential medium supplemented with 10% fetal calf serum (20). Cells
were subjected to hypoxia using an environmental chamber as described previously (17); pO2 in the medium was
12-14
torr. Cells subjected to hypoxia were placed in medium preequilibrated
with the hypoxic gas mixture just prior to placement in the
environmental chamber. Thus, cultures were immersed immediately in the
oxygen-deprived environment at the time of medium change/placement in
the chamber.
-actin as an internal control for RNA
loading. The same procedure was employed to assess Sp1 transcripts in
hypoxic murine lung, except 32P-labeled cDNA probe for
Sp1 was employed (22).
-actin IgG
(Sigma). Then, they were incubated with secondary antibody, an
affinity-purified peroxidase-conjugated IgG, either goat anti-rabbit
IgG or goat anti-rat IgG (Sigma).
424/
375 and
111/
65, respectively, from the murine Egr-1 promoter, linked to the basal thymidine kinase promoter (
80/+18) and the pGL3
basic vector (Promega). For pYSF63&64, site-directed mutagenesis was
employed to mutate GG to TT located at positions
414/
413 (pYSF63)
and
393/
392 (pYSF64), respectively, which flank a CArG box (SRE1)
(25-27). Cotransfection with pCMV-
-galactosidase was used as an
internal control for efficiency of transfection. Luciferase activity
was normalized based on
-galactosidase activity in the same well;
this is termed relative luciferase activity and is used in each of the
transfection studies.
II in Hypoxia-induced Expression of
Egr-1 and Tissue Factor in Cultured MPs--
Prior to experiments, the
medium bathing MPs was aspirated, and fresh medium containing 1% fetal
calf serum was added for 24 h. Cells were then placed in
serum-free medium with protein kinase C inhibitors, calphostin C,
GF109203X, or H-7 (all obtained from Sigma) for 60 min. Next, cultures
were transferred to the hypoxia chamber, and cells were harvested after
30 min (for detection of Egr-1 mRNA) or 4 h (for
detection of tissue factor mRNA).
111/+14]Luc; the latter spanned
111/+14 from
the tissue factor gene, including the hypoxia-responsive Egr-1 sites (19), linked to luciferase) (19, 28). Expression constructs for PKC isoforms were as follows: dominant-negative (DN)
PKC
(PKC
[K>R]) and DN-PKC
(PKC
[K>R]), as well as
constitutively active (CA) PKC
and CA-PKC
constructs (29);
CA-PKC
II (30, 31) and DN-PKC
II (M217), wild-type (wt) PKC
II
(rabbit, pcDNA130; mouse, pMTH
II); and wtPKC
I (rabbit,
pcDNA136) (32). The procedure for transfection utilized Superfect
Reagent, and the protocol was according to the manufacturer's
instructions. For transfection studies, macrophages were transferred to
medium with 1% fetal calf serum for 24 h. Then, cotransfection
was performed using either Egr-1 promoter-luciferase (1.5 µg; pYSF62) or tissue factor promoter-luciferase (2 µg;
pTF[
111/+14]Luc) constructs, one of the PKC-related constructs (1 µg), and pCMV-
-galactosidase (1 µg). Immediately after
transfection, cells were maintained in medium with 1% fetal calf serum
for 24 h and were then placed in serum-free medium for 20 h.
Cultures were next subjected to hypoxia for 4 h (for
Egr-1 studies) or 5 h (for tissue factor studies), and
luciferase activity was determined using a luminometer.
II was studied by immunoblotting
membranous fractions (33) from hypoxic U937 cells and lungs from mice
subjected to oxygen deprivation. Extracts were subjected to nonreduced
SDS-PAGE (7.5%) followed by Western blotting with rabbit anti-PKC
II
IgG (as primary antibody) and horseradish peroxidase-conjugated goat
anti-rabbit IgG (as secondary antibody; Amersham). Similar experiments
were performed with extracts of lung. Autophosphorylation of PKC
isoforms (PKC
I, PKC
II, and PKC
) was studied by labeling cultures with 32Pi as described (34, 35)
followed by immunoprecipitation with isoform-specific antibodies (Santa
Cruz Biotechnology), SDS-PAGE, and autoradiography. In brief, cultures
were maintained in normoxia, washed with HEPES-buffered phosphate-free
high glucose RPMI 1640, and labeled with 100 µCi of
32Pi for 3 h at 37 °C under normoxic
conditions. Then, cells were subjected to hypoxia for 5, 10, or 15 min
and processed as described (34, 35). Protein concentrations were
determined, and supernatants with the same amount of protein were
immunoprecipitated with antibodies to PKC
I, PKC
II, or PKC
(Santa Cruz Biotechnology).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
82 kDa (Fig. 1B). In contrast to the observed induction
of Egr-1, there was no increase in Sp1 in hypoxic lung,
either at the mRNA (Fig. 1A) or antigen level (Fig.
1C; two closely spaced bands corresponding to molecular masses of
95 and
105 kDa of similar intensity in normoxic and hypoxic lung were observed) (28). Immunohistochemistry revealed that
Egr-1 expression was not confined to a single cell type but that elevated amounts of Egr-1 antigen were present in a range of cells
in hypoxic lung (Fig. 1D) compared with normoxic controls (Fig. 1E). Analysis of adjacent sections using markers for
mononuclear phagocytes/macrophages (F4/80) (Fig. 1G) and
smooth muscle (smooth muscle
-actin IgG) (Fig. 1I)
demonstrated colocalization with increased Egr-1 (Fig. 1, F
and H). Closely paralleling enhanced levels of Egr-1, a
time-dependent appearance of Egr DNA binding activity was observed in nuclear extracts from hypoxic lung (Fig. 2, lanes 2-6).
Specificity of the DNA binding activity was shown by disappearance of
the gel shift band with excess unlabeled Egr probe (Fig. 2,
compare lanes 7 and 8), whereas oligonucleotides corresponding to sequences for AP-1, HIF-1, NF-IL-6, and Sp1 were without effect (Fig. 2, lanes 9-12). Furthermore, in a
previous study (19), we have found that the protein responsible for
Egr DNA binding activity in hypoxic lung was Egr-1, as shown
by complete disappearance of the band in the presence of anti-Egr-1 IgG
but not nonimmune IgG. These data indicate that Egr-1
expression and activation of Egr-1 DNA binding activity
occur in the lung within minutes of oxygen deprivation.
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Fig. 1.
Egr-1 expression in hypoxic murine
lung. Panel A, mice were subjected to hypoxia for the
indicated times followed by sacrifice and extraction of total RNA from
the lung. Northern analysis was performed with 32P-labeled
probes for Egr-1, Sp1, and -actin. In each case, 20 µg/lane of total RNA was loaded on the gel. Panels B and
C, immunoblotting was performed on nuclear extracts from the
lungs of mice subjected to hypoxia for 30 min using antibodies to Egr-1
(panel B) or Sp1 (panel C). In each case, 10 µg/lane of total protein was loaded on the gel. N and
H designate samples from normoxic and hypoxic lung,
respectively. Panels D-I, analysis of hypoxic lung for
Egr-1. Mice were exposed to hypoxia (panel D) or
normoxia (panel E) for 2 h, and lungs were prepared for
immunohistologic analysis using antibody to Egr-1. Adjacent sections
from hypoxic lung were stained with antibody to Egr-1 (panel
F) and F4/80 (panel G) or with antibody to Egr-1
(panel H) and smooth muscle
-actin (panel I).
The marker bar indicates 25 µm (panels D,
E, H, and I) and 8.3 µm
(panels F and G).
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Fig. 2.
Hypoxia enhances Egr DNA
binding activity in nuclear extracts prepared from murine lung.
Mice were subjected to hypoxia for the indicated times (lanes
3-6), nuclear extracts were prepared, and EMSA was performed with
32P-labeled Egr consensus probe. In other
experiments, mice were subjected to hypoxia for 30 min (lanes
7-12), nuclear extracts were prepared, and EMSA was performed.
Where indicated, nuclear extracts from hypoxic cultures were incubated
with 32P-labeled probe in the presence of a 100-fold molar
excess of unlabeled Egr, AP-1, HIF-1, NF-IL-6, or Sp1 probes
(lanes 8-12, respectively). FP indicates a lane
containing only 32P-labeled Egr free probe.
N and H denote samples from normoxic and hypoxic
lung, respectively.
Mechanisms of Hypoxia-induced Egr-1 Expression
The mechanisms were studied using a MP cell line as a model
system. Cultured MPs subjected to oxygen deprivation displayed time-dependent induction of Egr-1 mRNA (Fig.
3A) as well as increased Egr-1
antigen (Fig. 3C) and enhanced Egr DNA binding
activity in nuclear extracts (Fig. 3D). Maximal
Egr DNA binding activity by EMSA occurred within 30 min of
exposure to hypoxia, similar to what was observed in murine lung. The
rate of Egr-1 transcription was increased about 10-fold in
hypoxic MP cultures compared with normoxic controls (Fig.
3B).
|
Transient transfection of MPs with series of deletional/truncated
Egr-1 promoter-reporter (luciferase) constructs focused our
attention on the SRE motifs (Fig.
4A). As an internal control for efficiency of transfection, cotransfection studies were performed with pCMV--galactosidase. Results are shown as fold-increase in
luciferase activity normalized for
-galactosidase activity (termed
relative luciferase activity). The longest construct
(line A) displayed
5-7-fold increased
luciferase activity in hypoxia (this range was observed over four
experiments; results of a representative experiment are shown in the
figure). Truncation of AP-1 and Sp1 sites in the distal portion of the
promoter had virtually no effect on inducibility in hypoxia (Fig.
4A, line C). Consistent with their lack of
involvement, a construct spanning the latter elements (Fig.
4A, line B) linked to the most proximal portion
of the Egr-1 promoter did not drive luciferase expression in
hypoxia. The key portion of the promoter with respect to the effect of
hypoxia included the region spanning three SRE elements (Fig.
4A, line D). In contrast, neither of the proximal
SRE elements (Fig. 4A, lines E and F)
was able to confer enhanced expression of luciferase in response to
oxygen deprivation. In view of previous data implicating SRE DNA
binding motifs in hypoxic stress-associated up-regulation of GLUT1 and
studies of c-Fos expression (5, 41), we further analyzed two types of
SRE sites in the Egr-1 promoter, EBS-SRE-EBS (
424/
375
base pairs), from the distal portion of the promoter, and SRE-EBS-SRE
(
114/
65), from the proximal portion of the promoter. Transfection
studies were performed with constructs made by ligating EBS-SRE-EBS
(
424/
375) or SRE-EBS-SRE (
114/
65) to the basal thymidine kinase
promoter and luciferase, resulting in the vectors pYSF62 and pYSF65,
respectively (Fig. 4B). Only transfection with pYSF62
resulted in a hypoxia-mediated increase in luciferase activity; this is
consistent with the results in Fig. 4A, line D.
Furthermore, in EBS-SRE-EBS (
424/
375), mutational inactivation of
the proximal EBS (pYSF64) largely blocked hypoxia-induced expression,
whereas mutation of the distal EBS (pYSF63) completely prevented
hypoxia-inducibility of luciferase expression (Fig. 4B).
These data indicate the essential contribution of the EBS element in
the Egr-1 promoter for activation of transcription at the
SRE in response to hypoxia. Stimulated by these results, we performed
studies to assess a possible role for the ets factor Elk-1 and a kinase
cascade leading to its activation, in the up-regulation of
Egr-1 consequent to oxygen deprivation (see below).
|
Definition of the Hypoxia-induced Cascade Resulting in Activation of Egr-1 Transcription
Recent studies have demonstrated that reductive stress results in
phosphorylation of ERK 1/2 leading to activation of the ets factor
Elk-1(5, 42). The latter, in concert with serum response factor,
participates in initiation of Egr-1 gene transcription under
other experimental conditions (43). First, we tested whether hypoxia
triggered these events in hypoxic cultured macrophages, and then we
sought the basis for MAP kinase activation in our hypoxic cells.
Following induction of hypoxia, activation of Elk-1 occurred as
assessed using an Elk-1 chimeric reporter system in which
luciferase expression was monitored (39) in transfected MPs subjected
to oxygen deprivation (Fig.
5A; bar marked
pcDNA3, compare increased luciferase expression in
hypoxic versus normoxic cultures). The contribution of Elk-1
to transcriptional activation of Egr-1 was studied by
cotransfection of MPs with pYSF62, the Egr-1
promoter-luciferase construct encoding the hypoxia-responsive site
(Fig. 4B), and a construct encoding DN-Elk-1. In the
presence of DN-Elk-1, expression of the luciferase reporter was blocked compared with controls (Fig. 5B).
|
Role of PKCII--
Previous studies have shown that activation
of PKC stimulates expression of Egr-1 and tissue factor,
especially in epithelial cells (28). This led us to speculate that PKC
might be the proximal trigger in MPs leading to a kinase cascade,
including raf, MEK, and MAP kinases, which ultimately acts on Elk-1. To
test this hypothesis, experiments were performed to examine whether PKC might contribute to hypoxia-induced up-regulation of Egr-1
and tissue factor. MPs were exposed to 100 nM calphostin C,
10 µM GF109203X, or 20 µM H-7 and then
subjected to hypoxia. Total RNA was harvested after 30 min, and
Northern blotting was performed using radiolabeled mouse
Egr-1 cDNA. Hypoxia increased levels of Egr-1
mRNA in hypoxic MPs (Fig.
6A, lane 2), as
described above (Fig. 3). Cultures pretreated with PKC inhibitors
demonstrated suppression of Egr-1 mRNA in the hypoxic
environment (lanes 4, 6, 8); levels
were similar to that in normoxic controls (lane 1). Similar
results were obtained with respect to the effect of PKC inhibitors on
the level of tissue factor transcripts. MPs subjected to hypoxia
displayed increased levels of tissue factor transcripts compared with
controls maintained in normoxia (Fig. 6B, compare
lanes 9 and 10), as reported previously (19). In contrast, this increase in tissue factor mRNA was blocked in
cultures pretreated with PKC inhibitors (lanes 11-13).
Although these PKC inhibitors are not subtype-specific, they do, at the
concentrations employed, show relative selectivity for PKC
versus cAMP-dependent and
cGMP-dependent protein kinases. Thus, it seemed likely that the hypoxia-associated increase in Egr-1 and tissue factor
transcripts in MPs involved PKC.
|
PKCII is present in MPs and has been shown, in other cell types, to
participate in cellular responses to environmental perturbations, such
as increased levels of glucose (44, 45). Quiescent monocyte-like cells
(U937) subjected to hypoxia demonstrated an increase in PKC
II
antigen in the membranous fraction compared with normoxic controls
(Fig. 7A, lanes 1 and 2). Consistent with these observations in cell culture,
extracts from murine lung also demonstrated increased PKC
II antigen
in membranous fractions within
5-10 min of hypoxia compared with
normoxic controls (Fig. 7B, lanes 1-3). These
data suggested that PKC
II might become activated in response to
hypoxia, leading us to test this concept directly by assessing
autophosphorylation of PKC
II, the latter closely correlated with PKC
activation (35). In addition to PKC
II, our attention was focused on
PKC
I and PKC
. Both are expressed by MPs (44), although PKC
I
bears strong similarity to PKC
II (both are conventional PKCs and
represent alternatively spliced products of the same gene; PKC
represents a structurally distinct class of PKCs, the novel PKCs).
Cultured MPs subjected to oxygen deprivation were labeled with
32Pi and immunoprecipitated with antibodies for
PKC
I, PKC
II, or PKC
(Fig. 7C). Strong labeling of
PKC
II was observed following hypoxia (
4-fold increased intensity
of the immunoprecipitated band by 10 min), whereas there was virtually
no change in the intensity of the bands for PKC
I and PKC
.
|
These data led us to examine the effect of blocking PKCII by
overexpressing a dominant-negative construct. For these studies, we
monitored induction of Egr-1 transcription utilizing
transient transfection of MPs with the Egr-1
promoter-luciferase construct pYSF62 (Fig. 7D, left
panel). Cotransfection of MPs with both pYSF62 and the DN-PKC
II
expression vector (M217) blocked the hypoxia-associated increase in
luciferase activity observed in cultures transfected with the
Egr-1 promoter-luciferase construct alone and subjected to
hypoxia (Fig. 7D). In contrast, overexpression of constructs
for DN-PKC
or DN-PKC
in the same system had no effect on
luciferase expression after transfection of pYSF62 (Fig. 7D). Consistent with the specificity of the inhibition
caused by DN-PKC
II, transfection experiments with wtPKC
II (either
rabbit (r) or murine (m) in Fig. 7D)
in place of the DN-PKC
II construct showed no inhibitory effect. As a
further test for specificity of the observed results with DN-PKC
II,
"rescue" experiments were performed by cotransfecting MPs with
DN-PKC
II + wtPKC
II or DN-PKC
II + wtPKC
I, and determining
the effect on expression of pYSF62 under hypoxic conditions (Fig.
7D). Using these reagents, it has been shown previously that
overexpression of wtPKC
II, but not PKC
I, will reverse the
phenotype induced by transfection with DN-PKC
II in a skeletal muscle
cell culture system (32). In our studies, wtPKC
II, but not
wtPKC
I, restored expression of pYSF62 under hypoxic conditions in
the presence of DN-PKC
II (Fig. 7D). Additional support
for a central role of PKC
II in the expression of Egr-1 by
MPs was shown by increased expression of the luciferase reporter after
cotransfection of constitutively active PKC
II along with pYSF62,
even when cells were maintained in normoxia (Fig. 7D,
right panel). As expected, cotransfection of cultures with
constructs encoding CA-PKC
or CA-PKC
also induced expression of
pYSF62 under normoxic conditions, consistent with the well known
ability of diverse PKC isoforms to activate downstream effector mechanisms, such as MAP kinases and Egr-1. These data are
consistent with an important contribution for activation of PKC
II in
hypoxic MPs as a trigger for increased transcription of
Egr-1. Specificity appears to occur at the level of
preferential PKC
II activation in hypoxic MPs versus
apparent lack of comparable PKC
I or PKC
activation.
Inhibition of PKCII also suppressed activation of tissue factor
transcription in hypoxic MPs. Expression of tissue factor was monitored
using the tissue factor promoter-luciferase construct spanning the
hypoxia responsive Egr-1 elements (pTF[
111/+14]Luc; Fig.
7E) (39). Although hypoxia caused increased luciferase activity/expression with the tissue factor promoter-luciferase construct alone (pTF[
111/+14]Luc), cotransfection with the
DN-PKC
II suppressed luciferase activity (Fig. 7E). In
contrast, neither mock-transfected controls nor transfection with
constructs encoding DN-PKC
or DN-PKC
, in place of DN-PKC
II,
demonstrated an inhibitory effect (Fig. 7E). Furthermore,
transfection of MPs with wtPKC
II in place of the DN-PKC
II
construct did not cause a suppression of luciferase activity due to
pTF[
111/+14]Luc in hypoxia. The importance of PKC
II in
regulating tissue factor expression was emphasized by the results of
studies in which CA-PKC
II was overexpressed; luciferase activity
with pTF[
111/+14]Luc increased even when cells were maintained in
normoxia (Fig. 7E, right panel). Consistent with
the ability of multiple PKC isoforms to activate downstream targets,
increased expression of pTF[
111/+14]Luc was also observed after
cotransfection with constructs encoding CA-PKC
or CA-PKC
. As
indicated above, these data suggest that specificity for involvement of
PKC
II in the cascade leading to Egr-1 and tissue factor
transcription consequent to oxygen deprivation occurs at the level of
preferential hypoxia-mediated activation of this PKC isoform.
Role of Raf, MEK, and MAP Kinases--
Studies were performed to
link activation of PKCII to activation of MAP kinases and Elk-1 in
MPs subjected to oxygen deprivation. Activation of ERK 1/2 occurred
within 10 min of subjecting MPs to hypoxia, based on an in-gel kinase
assay (Fig. 8A). Densitometry demonstrated an
5-7-fold increase in the intensity of the
immunoreactive ERK 1/2 bands after oxygen deprivation (comparing band
intensity in normoxia (N) and hypoxia (H) 5 min).
The central role of ERK 1/2 in activation of Egr-1
transcription was shown using constructs encoding DN-ERK 1 and DN-ERK
2. Cotransfection of MPs with pYSF62 and DN-ERK 1 or DN-ERK 2 suppressed hypoxia-induced expression of the luciferase reporter (Fig.
8C), consistent with the possibility that either DN form
blocked interaction of ERKs with their downstream targets. These
results suggested that MEK 1/2 might also be involved in the pathway
leading to hypoxia-mediated Egr-1 transcription. Addition of
the MEK inhibitor PD98059 to hypoxic MPs completely suppressed
Egr-1 transcripts (Fig. 8B). Consistent with
these results, hypoxia-mediated ERK 1/2 activation, assessed by in-gel kinase assay, was suppressed in the presence of PD98059 (Fig. 8A). At the concentration employed (10 µM),
the MEK inhibitor is relatively selective for MEK 1 (IC50
1-3 µM), although it may also have a lesser effect on
MEK 2 (IC50
50 µM) (37).
|
Next, we sought to define a possible role for Raf in events leading to Elk-1 and Egr-1 activation. Cotransfection of MPs with DN-raf and pYSF62 prevented increased transcription of Egr-1, based on evaluating luciferase activity after exposure of cells to hypoxia (Fig. 8D).
It was important to relate activation of Elk-1 in hypoxic MPs to each
of the steps examined above. The GAL4 system was used to assess Elk-1
activation (46), along with transfection of one of the following
constructs: either DN-PKCII, DN-raf, DN-ERK 1, DN-ERK 2, or
pcDNA3 alone. After transfection, MPs were transferred to the
hypoxia chamber, and expression of luciferase activity was monitored
subsequently. In each case, the dominant-negative construct blocked
expression of luciferase activity, whereas pcDNA3 alone was without
effect (Fig. 5A). The GAL4-Elk-1-luciferase expression
system was tested further by transfecting cultured MPs with
CA-PKC
II; under these conditions, increased luciferase activity was
observed in normoxia (Fig. 5C). As expected, overexpression of CA-PKC
or CA-PKC
in normoxic MPs also resulted in increased Elk-1-luciferase activity. These data are consistent with the results
described above (related to Fig. 7E), in which each of the
constitutively active PKC isoforms triggered events leading to
Egr-1 transcription, although specificity with respect to
hypoxic stress appeared to result from selective activation of
PKC
II in oxygen-deprived MPs.
Relationship of Egr-1 and HIF-1 in Hypoxia
Together with HIF-1, ARNT/HIF-1
forms the HIF-1 DNA-binding
complex implicated in up-regulation of multiple genes in response to
oxygen deprivation (1, 3). Possible relationships between Egr-1 and HIF-1 activation were examined using mutant mouse
hepatoma cells deficient in ARNT/HIF-1
termed C4 cells (in the
figure denoted as Mut) and comparing results with the
wild-type counterpart from which they were derived, Hepa1c1c7 cells
(20). Northern analysis demonstrated comparable hypoxia-mediated
increase in Egr-1 transcripts in wild-type (Hepa1c1c7) and
Mut (C4) hepatoma cells (Fig.
9A, lanes 2,
4, 6, and 8) compared with normoxia
(Fig. 9A, lanes 1, 3, 5,
and 7). By gel shift analysis with 32P-labeled
Egr probe, nuclear extracts derived from hypoxic wild-type hepatoma cells showed a prominent gel shift band compared with normoxic
controls (Fig. 9B, lanes 10 and 11). A
similar increase in the Egr gel shift band was observed with
nuclear extracts from mutant C4 cells (Fig. 9B, lanes
12 and 13). Thus, the HIF-1- and Egr-dependent pathways appear to be initiated
independently.
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DISCUSSION |
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Our work on hypoxia-associated induction of MP tissue factor,
leading to vascular fibrin deposition, assigns activation of PKCII
and Egr-1 a pivotal role in the cellular response to oxygen deprivation. We have outlined a pathway in MPs whereby activation of
PKC
II occurs within minutes of hypoxia and triggers events leading
to activation of Elk-1, eventuating in transcription of Egr-1. Increased levels of Egr-1 are evident
in vivo in hypoxic lung, especially in MPs, and in cultured
MPs subjected to oxygen depletion. Egr-1 expression under
hypoxic conditions is closely tied to its activation, as assessed by
induction of Egr DNA binding activity, with subsequent
transcription of the tissue factor gene mediated by Egr
motifs present in the serum response region of the promoter as a trio
of overlapping Sp1/Egr DNA-binding elements (19, 28).
Expression of tissue factor enables hypoxic monocytic cells to trigger
the procoagulant pathway resulting in fibrin formation. We propose that
this sequence of events, initiated virtually at the onset of hypoxic
stress, sets the stage for vascular dysfunction by providing a nidus
for generation of procoagulants and fibrin. The most far reaching
implication of these results concerns the potential of activated
PKC
II and Egr-1 in monocytes to recruit multiple
downstream cellular targets thereby engaging a range of cellular
effector mechanisms. Thus, induction tissue factor, presumably,
represents just one of many possible cellular responses modulated by
this pathway. Nonetheless, in view of the importance of tissue factor
for initiating coagulation, its expression in the intravascular space
is of significance and serves as an appropriate starting point for
analyzing roles of PKC
II and Egr-1 in ischemic stress.
There are intriguing parallels between our results concerning
Egr-1 expression and activation using hypoxia as the
stimulus and observations of others concerning the role of
Egr-1 in the cellular response to urea and shear stress (23, 47).
Hypoxia-mediated activation of transcription via Elk-1 in MPs,
presumably in complex with serum response factor (43), is analogous to
previous observations in HeLa cells concerning expression of
c-fos (5, 42). This pathway of hypoxia-associated induction of transcription at SREs may also explain, in part,
SRE-dependent induction of the
noninsulin-dependent glucose transporter (GLUT1) associated
with oxygen deprivation and inhibitors of aerobic respiration (41). In
the latter case, hypoxic induction of GLUT1 resulted from two
interactive mechanisms: direct effects of oxygen deprivation, due to an
HIF-1 DNA binding motif, and a response to inhibition of mitochondrial
respiration, mediated by sequences within 100 nucleotides 5' of the
HIF-1 site, which contained an SRE. These considerations suggest that
the PKCII/Egr-1 pathway under study in our work is most
likely a hypoxic stress-associated pathway rather than a direct
response to a change in ambient oxygen levels, the latter an apparent
property of HIF-1
(1). However, the capacity of cellular mechanisms
activated by PKC
II/Egr-1 to modulate cellular behavior
emphasizes their relevance to the host response to ischemic challenge.
In the setting of ischemia, much attention has been focused on the
contribution of PKC isoforms to preconditioning of the myocardium and
acute myocardial injury, although their role remains controversial (48,
49). Changes in PKC activity and isoform-specific increases in PKC in
the membrane fraction have also been noted during ischemic
neurodegeneration in the cerebellum and hippocampus in a canine model
(50, 51). Fewer experiments have addressed the role of PKCII in
ischemia, although a recent study has employed the PKC
inhibitor
LY333531 in a porcine model of ischemia-induced preretinal
neovascularization (52). Administration of the PKC
inhibitor
suppressed neovascularization, consistent with a contribution of PKC
to cellular events underlying angiogenesis. In this context, tissue
factor expression (shown to be induced by hypoxia in our studies) (19)
has been associated with angiogenesis, especially in tumor
neovasculature (53-55), and it is possible that hypoxia-regulated PKC
activation provides a link between these two events. However, most attention with regard to participation of PKC
II in vascular pathology has focused on cultured endothelial cells exposed to high
levels of ambient glucose and implications of the observed PKC
II
activation for vascular complications in diabetes (45). Our study in
cultured MPs adds a new facet to the biology of PKC
II in the setting
of oxygen deprivation: activation of PKC
II causes subsequent
expression of Egr-1 and, downstream, induction of tissue factor.
Egr-1 is an ubiquitous transcription factor previously
ascribed roles in a range of physiologic and pathophysiologic processes (26). With the generation of Egr-1 knockout mice, the
biologic contexts of Egr-1 function have been redefined
(56). Egr-1 null mice developed normally, and the only
defect noted early on was infertility in females caused by to failure
to produce luteinizing hormone-releasing hormone (56). Subsequently, we
demonstrated that expression of tissue factor and fibrin deposition in
response to hypoxia were severely blunted in Egr-1 null
mice, indicating a role for this transcription factor in ischemic
stress (19). Previous studies have shown Egr-1 activation in
other ischemic situations, such as renal and cardiac
ischemia/reperfusion (57, 58), and it is tempting to speculate that
induction of tissue factor in monocytes subjected to local hypoxemia
might magnify the ischemic response. Because increased expression and
activation of Egr-1 in response to oxygen deprivation
occurred independently of HIF-1, as shown by our experiments in
hepatoma cells deficient in HIF-1 function,
Egr-1-mediated effects in ischemia may drive other facets of
the host response. An important level of Egr-1 regulation
in vivo concerns its apparent selective up-regulation in
cellular elements in hypoxic lung; whereas MPs and smooth muscle cells
showed increased Egr-1 antigen, endothelial cells, for example, did not.
Expression of monocyte tissue factor is a final common pathway for
initiation of procoagulant events in the intravascular space (59).
Egr-1 is known to participate in the induction of tissue
factor, especially in epithelial-like cells and vascular smooth muscle
(28). Induction of tissue factor has also been shown previously to
involve PKC, based on studies with phorbol esters and general PKC
inhibitors (59). This is the first report, to our knowledge, linking
hypoxia to activation of PKCII, induction of Egr-1, and
expression of tissue factor in MPs. Although activation of PKC
II and
Egr-1 is clearly not specific for hypoxia-associated cell
stress, by recruiting multiple cellular effector mechanisms, this
pathway may have considerable impact on the outcome of ischemic events.
Further studies will be required to determine the contribution and
consequences of the PKC
II/Egr-1 pathway in hypoxia and ischemia.
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ACKNOWLEDGEMENTS |
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We acknowledge the technical assistance of Drs. Yun Gao and Chao Zhai in preliminary studies for this work.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grants HL42507, PERC, HL59488, and HL55397 and by the Surgical Research Fund.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.
a To whom correspondence should be addressed: Dept. of Surgery, P&S 11-420, College of Physicians and Surgeons of Columbia University, 630 West 168th St., New York, NY 10032.
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ABBREVIATIONS |
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The abbreviations used are: HIF-1, hypoxia-inducible factor-1; ARNT, arylhydrocarbon-receptor nuclear translocator; CA, constitutively active; CMV, cytomegalovirus; DN, dominant-negative; EBS, ets binding site; Egr, early growth response; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated protein kinase; IL-6, interleukin-6; MAP kinase, mitogen-activated protein kinase; MEK kinase, mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase kinase; MP, mononuclear phagocyte; NF, nuclear factor; PAGE, polyacrylamide gel electrophoresis; PKC, protein kinase C; SRE, serum response element; wt, wild-type.
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REFERENCES |
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