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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3300-3307
Hypoxia Stimulates Urokinase Receptor Expression Through a Heme
Protein-Dependent Pathway
By
Charles H. Graham,
Tania E. Fitzpatrick, and
Keith R. McCrae
From the Department of Anatomy and Cell Biology, Queen's University,
Kingston, Ontario, Canada; and the Sol Sherry Thrombosis Research
Center, Temple University School of Medicine, Philadelphia, PA.
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ABSTRACT |
Hypoxia underlies a number of biologic processes in which cellular
migration and invasion occur. Because earlier studies have shown that
the receptor for urokinase-type plasminogen activator (uPAR) may
facilitate such events, we studied the effect of hypoxia on the
expression of uPAR by first trimester human trophoblasts (HTR-8/SVneo)
and human umbilical vein endothelial cells (HUVEC). Compared with
control cells cultured under standard conditions (20% O2),
HTR-8/SVneo cells and HUVEC cultured in 1% O2 expressed more uPAR, as determined by flow cytometric and
[125I]-prourokinase ligand binding analyses. Increased
uPAR expression paralleled increases in uPAR mRNA. The involvement of a
heme protein in the hypoxia-induced expression of uPAR was suggested by
the observations that culture of cells with cobalt chloride, or sodium 4,5-dihydroxybenzene-1,3-disulfonate (Tiron), an iron-chelating agent,
also stimulated uPAR expression, and that the hypoxia-induced uPAR
expression was inhibited by adding carbon monoxide to the hypoxic
atmosphere. Culture of HTR-8/SVneo cells with vascular endothelial
growth factor (VEGF) did not increase uPAR mRNA levels, suggesting that
the hypoxia-mediated effect on uPAR expression by these cells did not
occur through a VEGF-dependent mechanism. The functional importance of
these findings is suggested by the fact that HTR-8/SVneo cells cultured
under hypoxia displayed higher levels of cell surface plasminogen
activator activity and greater invasion through a reconstituted
basement membrane. These results suggest that hypoxia may promote
cellular invasion by stimulating the expression of uPAR through a heme
protein-dependent pathway.
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INTRODUCTION |
HYPOXIC STRESS UNDERLIES a number of
biologically-important processes in which cellular migration and
invasion occur. For example, hypoxia within an expanding tumor leads to
the release of vascular endothelial growth factor (VEGF) and
stimulation of angiogenesis,1 the success of which depends
on endothelial cell migration and invasion. Hypoxia may also play an
important role in promoting tumor metastasis and invasion of the
extracellular matrix.2-5 It is likely that specific
responses to hypoxia also promote cellular migration during processes
such as atherosclerotic plaque development, as suggested by the
expression of hypoxia-inducible proteins by plaque
macrophages,6 as well as during early human pregnancy,
where blastocyst implantation and uterine invasion by trophoblast cells
occur under conditions of relative hypoxia.7,8
The expression of pericellular plasminogen activator activity may
facilitate the cellular migration and invasion that occur in the above
settings.9 Generation of such activity is dependent on the
binding of urokinase-type plasminogen activator (uPA) to the urokinase
receptor (uPAR), a 55-kD glycoprotein anchored to the
plasma membrane by a glycosyl-phosphatidylinositol
moiety.10 The urokinase receptor promotes cellular
migration through several mechanisms, one of which involves its ability
to amplify and focus the expression of plasminogen activator activity
to discrete sites on the cell surface.11,12 In addition,
the uPAR mediates cellular adhesion to vitronectin,13
promotes integrin-dependent migration,14 and initiates
intracellular signaling events.15
In light of the above considerations, we have investigated whether
hypoxia plays a role in regulating the expression of uPAR by invasive
first trimester human trophoblasts and human umbilical vein endothelial cells (HUVEC). Our studies suggest that hypoxia stimulates uPAR expression and cellular invasion through a
reconstituted extracellular matrix and that these effects are mediated
through an oxygen-binding heme protein.
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MATERIALS AND METHODS |
Materials.
Tissue culture medium was obtained from GIBCO-BRL (Grand Island, NY).
Fetal bovine serum (FBS) was from Hyclone (Logan, UT) or from
GIBCO-BRL. Cobalt chloride and 4,5-dihydroxybenzene-1,3-disulfonate (Tiron) were purchased from Sigma Chemical Co (St Louis, MO). Control
mouse IgG2a and fluorescein isothiocyanate
(FITC)-conjugated secondary antibodies were obtained from Dako Corp
(Carpinteria, CA). The fluorogenic plasmin peptide substrate,
HDVALL-AMC, was purchased from Bachem (King of Prussia, PA). Urokinase
enzyme-linked immunosorbent assay (ELISA) kits, plasminogen, and the
monoclonal anti-uPAR antibody used to measure uPAR expression by flow
cytometry (monoclonal antibody [MoAb] 3937), were obtained from
American Diagnostica, Inc (Greenwich, CT). Prourokinase was a generous gift from Drs Jack Henkin and Andrew Mazar (Abbott Laboratories, Abbott
Park, IL). Matrigel was obtained from Collaborative Biomedical Products
(Bedford, MA), and invasion chambers (8.0 µmol/L pore size) from
Costar Corp (Toronto, Canada). Endothelial cell growth supplement was
purified as described by Maciag et al.16 Recombinant VEGF
was purchased from R & D Systems (Minneapolis, MN). Radiochemicals and
Reflection NEF autoradiographic film were from Dupont/New England
Nuclear (Mississauga, Canada), and Pharmacia Oligolabelling kits were
obtained from Pharmacia Biotech (Piscataway, NJ). Iodobeads were
purchased from Pierce (Rockford, IL). Nylon membranes were from Micron
Separations, Inc (Westboro, MA). Airtight chambers used for culture of
cells under hypoxic conditions were obtained from BellCo Biotechnology
(Vineland, NJ).
Cells.
HTR-8/SVneo cells were obtained from explant cultures of human first
trimester placenta and immortalized by transfection with a cDNA
construct containing the SV40 large T antigen.17 These cells have been previously characterized17 and have been
maintained in culture for more than 120 passages in RPMI 1640 supplemented with 5% FBS. They exhibit a high proliferation index and
share various phenotypic similarities with the nontransfected parent HTR-8 cells such as in vitro invasive ability and lack of
tumorigenicity in nude mice.17 HUVEC were isolated from
pooled umbilical cords as previously described18 and
cultured in Medium 199 containing 75 µg/mL endothelial cell growth
supplement, 2 mmol/L glutamine, and 5% FBS.
Culture conditions to assess the cellular response to hypoxia.
For culture under hypoxic conditions, cells were placed in airtight
chambers, which were flushed with a gas mixture containing 5%
CO2 and 95% N2 until the oxygen concentration
within the chamber, measured using a Miniox 1 oxygen analyzer (Catalyst
Research Corp, Owings Mills, MD), was less than 0.5%. Cells were then
incubated within the sealed chambers for up to 24 hours, at 37°C.
Under these conditions, the O2 concentration equilibrates
within 1 to 2 hours and remains at or below 1% throughout the
incubation period.
Binding of molecular oxygen to the ferrous iron atom within the
porphyrin ring of a heme protein induces a change in its conformation from the deoxy, or tense, to the oxy, or relaxed state, as described for hemoglobin.19 The conformation of a putative
oxygen-sensing heme protein may be regulated in a similar manner, and
such a protein may acquire signaling activity after assuming the deoxy state, as described for the FixL heme protein of Rhizobium
meliloti.20 To determine whether the pathway underlying
the regulation of uPAR by hypoxia involves such a protein, we cultured
cells for 24 hours in the presence or absence of either 100 µmol/L
cobalt chloride or 30 mmol/L Tiron,21 an iron-chelating
agent. Cobalt displaces iron from the porphyrin ring, but binds oxygen
with lower affinity than iron,22 while Tiron chelates
intracellular iron in a manner similar to that described for
desferrioxamine.23 Hence, culture of cells in the presence
of either of these agents induces the conformation of heme proteins
into the deoxy state, thereby initiating cellular hypoxic responses.
Furthermore, like oxygen, carbon monoxide also binds to heme proteins,
maintaining them in the oxy or relaxed state, even under hypoxic
conditions. Therefore, another strategy used to assess the involvement
of such a protein in regulation of uPAR expression by hypoxia was to
culture HTR-8/SVneo cells for 24 hours under hypoxic conditions in the
presence of 20% carbon monoxide, with the expectation that inclusion
of the latter would block heme protein-mediated hypoxic responses.
Determination of uPAR expression by flow cytometry and urokinase
binding analysis.
For flow cytometry, cells were released from flasks by incubation in
cold phosphate-buffered saline (PBS) containing 5 mmol/L EDTA. One
million cells were then incubated for 1 hour at 4°C, with either 10 µg/mL of MoAb 3937, or control mouse IgG2a, used at the same
concentration. Bound antibody was detected using FITC-conjugated goat
anti-mouse immunoglobulin. Cells were then fixed with 2% paraformaldehyde in PBS before analysis using a Coulter Elite Flow
Cytometer (Beckman Coulter Instruments, (Miami, FL).
Measurement of uPAR expression by ligand binding analysis was performed
using [125I]-prourokinase as the ligand, as we have
described previously.24 Prourokinase was radiolabeled with
125I, using Iodobeads, to a specific radioactivity of at
least 106 cpm/µg. Cells were then chilled to 4°C,
washed with cold PBS containing 1% bovine serum albumin (BSA), and
incubated for 2 hours with increasing concentrations of
[125I]-prourokinase in the absence (to determine total
binding) or presence (to determine nonspecific binding) of a 100-fold
molar excess of unlabeled prourokinase. Specific binding was defined as
the difference between total and nonspecific binding and analyzed by
nonlinear curve fitting methods (least squares method) using the
Kaliedograph software program (Synergy Software, Reading, PA).
Northern blot analysis.
Total cellular RNA was isolated, separated by electrophoresis, and
transferred to charged nylon membranes. After prehybridization at
42°C for 2 hours in 50% formamide, 5× Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), 6× SSC (1× SSC = 0.15 mol/L NaCl, 15 mmol/L sodium citrate, pH 7.0) and 100 µg/mL denatured salmon sperm DNA, membranes were hybridized overnight at 42°C with
a full-length uPAR cDNA probe12 cloned in a Bluescript vector and labeled with [ -32P] deoxycytidine
5 -triphosphate (dCTP) using a Pharmacia Oligolabelling kit. The hybridization solution consisted of 6× SSC, 0.5% SDS, 100 µg/mL denatured salmon sperm DNA and 50% formamide. After serial
washes, the membrane was developed by autoradiography using Dupont
Reflection NEF film. To determine whether VEGF plays a role in uPAR
upregulation in HTR-8/SVneo cells, as shown previously for endothelial
cells,25 relative levels of uPAR mRNA in cells cultured for
24 hours under either 20% O2 alone, 1% O2, or
20% O2 + 10 ng/mL VEGF were also assessed by Northern blot
analysis.
In vitro invasion assay.
A previously-described invasion assay was used to determine the effect
of hypoxia on the invasion of HTR-8/SVneo cells through a reconstituted
basement membrane.17 Briefly, Transwell invasion chambers
were coated with 100µL of a 1.3 mg/mL solution of Matrigel diluted in
cold RPMI 1640 medium. The Matrigel was then air-dried for 12 hours in
a laminar flow cabinet. HTR-8/SVneo cells were labeled by incubation
for 24 hours in the presence of 10 µCi/mL [3H]-thymidine. The cells were then harvested, adjusted
to a concentration of 5.0 × 105/mL, and 100-µL
aliquots added in triplicate to the upper wells of the invasion
chambers. After a 24-hour incubation, cells in the upper and lower
compartments of the chambers were harvested.17 The
invasion index, reflecting the percentage of added cells that had
invaded the Matrigel was determined by measurement of the radioactivity
in the upper and lower compartments, as well as in the
membrane.17
Determination of plasminogen activator (PA) and gelatinase levels in
the culture medium.
Concentrations of gelatinase and plasminogen activators in the
conditioned medium of HTR-8/SVneo cells cultured under standard or
hypoxic conditions were compared using gel zymography, as previously described.17 The concentrations of uPA antigen in these
samples were more accurately measured using a specific uPA ELISA.
Determination of cell surface PA activity.
Cell surface PA activity was determined using a modification of the
fluorometric assay of Ellis et al.26 Briefly, HTR-8/SVneo cells were plated in quadruplicate wells of a 96-well tissue culture plate and allowed to grow to 95% confluency. After a 24-hour
incubation under an atmosphere of either 20% O2 or 1%
O2, the cells were washed twice and further incubated with
fresh medium containing plasminogen and the plasmin peptide substrate,
HDVLL-AMC, used at concentrations of 0.2 µmol/L and 0.5 mmol/L,
respectively. Plasmin generation was assessed by determining the
fluorescence within individual microplate wells 30 minutes later, using
a Perkin-Elmer LS50B Luminescence Spectrophotometer (Perkin-Elmer Corp,
Norwalk, CT) (excitation wavelength 360 nm, emission wavelength 460 nm).
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RESULTS |
Urokinase receptor expression is stimulated by hypoxia.
We first determined the effect of hypoxia on the expression of uPAR by
HTR-8/SVneo cells (Fig 1A). In comparison
to cells cultured under standard conditions (20% O2), the
expression of uPAR by cells cultured in 1% O2 for 24 hours
was 68% higher (n = 7, P = .007), as determined by flow
cytometry. Equal amounts of nonimmune mouse IgG2a bound to
cells cultured under both standard and hypoxic conditions,
demonstrating that increased binding of anti-uPAR MoAb 3937 was not due
to nonspecific interactions or increased Fc receptor expression.
Similarly, assessment of uPAR expression by HTR-8/SVneo cells through
measurement of [125I]-prourokinase binding showed that
hypoxia stimulated the expression of uPAR by 88% (n = 9, P = .003), an increment similar to that detected using flow cytometry. This
was accompanied by a parallel increase in the kd
(Table 1), as well as by an increase in the cellular content of uPAR mRNA (Fig 1B). Increased uPAR mRNA expression was first apparent after 4 hours of exposure to hypoxia and reached a
maximum level after 6 hours.
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| Fig 1.
Effect of hypoxia on uPAR protein and mRNA levels in
HTR-8/SVneo cells. (A) Analysis of uPAR expression by HTR-8/SVneo cells using flow cytometry showed an average increase of 68% in the mean
fluorescence intensity when cells were cultured under hypoxic conditions and labeled with MoAb 3937. This figure is representative of
seven independent experiments. (B) Northern blot analysis showed a
2.5-fold increase in uPAR mRNA levels after only 4 hours of hypoxic
culture when compared with uPAR mRNA levels in cells cultured under
standard conditions. The levels of uPAR transcript increased by
fivefold at 6 hours of culture under hypoxia and remained high at 8 and
24 hours. Relative levels of uPAR mRNA were determined with a SigmaGel
gel analysis program using 28S rRNA to correct for differences in the
amount of RNA loaded onto the gel.
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Table 1.
Effects of Hypoxia, Tiron, and Cobalt Chloride on the
Binding of [125I]-Prourokinase to HTR-8/SVneo Cells
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It has been previously reported that VEGF increases the expression of
uPAR by vascular endothelial cells.25 Furthermore, recent
studies have shown that hypoxia stimulates VEGF release by increasing
transcriptional activation of the VEGF gene via hypoxia-inducible factor-1 (HIF-1), as well as through mRNA
stabilization.27-30 In the present study, however, culture
of HTR-8/SVneo cells in the presence of 10 ng/mL of VEGF for 24 hours
under normoxic conditions did not result in increased levels of uPAR
transcript (Fig 2), suggesting that the
hypoxia-mediated upregulation of uPAR occurs through a VEGF-independent
mechanism.
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| Fig 2.
Effects of hypoxia and VEGF on uPAR mRNA levels in
HTR-8/SVneo cells assessed by Northern blot analysis. VEGF did not
increase the expression of uPAR mRNA when incubated with cells under
normoxic conditions.
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We were also interested in determining whether the effects of hypoxia
on uPAR expression extended to other cell types such as endothelial
cells. Figure 3 shows results in which
similar effects of hypoxia on upregulation of uPAR expression were
detected by radioligand binding studies using HUVEC. These results were also confirmed by flow cytometry (not shown).
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| Fig 3.
Binding of [125I]-prourokinase to HUVEC,
cultured under standard (solid squares) and hypoxic (open triangles)
conditions. This figure is representative of three independent
experiments in which a mean increase of 46% in the binding of
[125I]-prourokinase to cells cultured under hypoxic
conditions was observed. Error bars indicate standard error of
triplicate samples for each point.
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Role of a heme protein in the regulation of uPAR expression.
To examine the potential involvement of a heme protein in the
hypoxia-mediated stimulation of uPAR expression, we first examined the
expression of uPAR by HTR-8/SVneo cells in response to incubation in
the presence of cobalt chloride or Tiron. Both flow cytometric and
radioligand binding studies showed a threefold increase in the
expression of uPAR in response to Tiron (n = 6, P = .004), with
a more modest 28% increase observed in response to cobalt chloride (n = 8, P = .014; Table 1). In each case, the kd increased as
well, as observed following exposure of cells to hypoxia. Stimulation of uPAR expression by either cobalt or Tiron was associated with increased levels of uPAR mRNA, comparable to those observed in response
to hypoxia (Fig 4).
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| Fig 4.
Effect of hypoxia, carbon monoxide, cobalt chloride, and
Tiron on the levels of uPAR mRNA in HTR-8/SVneo cells. Cells were cultured for 24 hours under the conditions listed over each lane of the
figure. Levels of uPAR mRNA were increased 2.8, 1.8, 3.5, and
>10-fold, respectively, within cells cultured under hypoxic (1%
O2) conditions, hypoxic conditions in the presence of 20% carbon monoxide, and standard (20% O2) conditions in the
presence of either cobalt chloride or Tiron, in comparison to cells
cultured under standard conditions alone (lane 1). Carbon monoxide
reduced the hypoxia-mediated increase in uPAR mRNA by 35%.
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To further assess the potential role of a heme protein in the increased
expression of uPAR in response to hypoxia, we examined the effects of
carbon monoxide on this response. As expected, increased uPAR
expression after culture of HTR-8/SVneo cells under hypoxic (1%
oxygen) conditions was inhibited (56%) by inclusion of 20% carbon
monoxide in the gas mixture (Fig 5); a
parallel decrease was observed in the content of cellular uPAR mRNA
(Fig 4). Furthermore, in two independent experiments, the inhibitory effects of carbon monoxide on the hypoxia-induced expression of uPAR
were completely prevented by inclusion of cobalt chloride or Tiron in
the medium (not shown), indicating that the inhibition of uPAR
expression by carbon monoxide under hypoxic conditions was
not due to nonspecific toxicity.
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| Fig 5.
Inhibition of hypoxia-induced uPAR expression by carbon
monoxide. HTR-8/SVneo cells were incubated for 24 hours under standard (20% O2) or hypoxic (1% O2) conditions, or
hypoxic conditions in the presence of 20% carbon monoxide. The
expression of uPAR was then assessed by flow cytometry using MoAb 3937. Carbon monoxide inhibited the hypoxia-induced expression of uPAR by
56%. Data represent the mean ± standard error of four independent
experiments.
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Hypoxia stimulates in vitro invasiveness via a heme protein.
To determine the functional correlates of hypoxia-induced uPAR
expression, we compared the invasion of HTR-8/SVneo cells through a
reconstituted basement membrane (Matrigel) under both standard (20%
oxygen) and hypoxic (1% oxygen) conditions, observing an increase of
41.4% ± 7.4% (P = .003) under the latter
(Table 2). Similar increases were observed
when invasion assays were performed under 20% oxygen in the presence
of either 100 µmol/L cobalt chloride (24.6 ± 8.4%;
P = .028) or 30 mmol/L Tiron (29.3 ± 12.7%;
P = .035) (Table 2). The role of a heme protein in the
regulation of cellular invasiveness was confirmed by experiments in
which hypoxia-stimulated invasion was inhibited by 87% in the presence of 20% carbon monoxide and was not significantly different from that
which occurred in the presence of 20% oxygen (Table 2).
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Table 2.
Effects of Hypoxia, Carbon Monoxide, Tiron, and Cobalt
Chloride on Invasion of HTR-8/SVneo Cells Through Matrigel
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Effect of hypoxia on PA and gelatinase levels in culture medium and
on cell surface PA activity.
Gel zymography showed a time-dependent reduction in the levels of PAs
in medium conditioned by HTR-8/SVneo cells cultured for up to 24 hours
under 1% O2 (Fig 6). These
observations were supported by additional studies in which direct
measurement of urokinase antigen levels in the conditioned medium of
HTR-8/SVneo cells cultured under hypoxic conditions for 24 hours were
reduced by a mean of 52%. In contrast, the expression of cell surface PA activity by HTR-8/SVneo cells cultured for 24 hours under hypoxic conditions was 20% higher than that expressed by cells cultured under
standard conditions (P = .000007). These findings are
consistent with binding of secreted urokinase to increased numbers of
cellular uPAR.
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| Fig 6.
Zymographic analysis of PAs present in the medium of
HTR-8/SVneo cell cultures incubated for 8, 12, and 24 hours under
normoxic or hypoxic conditions. Casein and plasminogen were
incorporated into the acrylamide before polymerization as detailed
elsewhere.17 Samples of serum-free medium containing 200 ng
of protein were loaded onto each lane and separated by electrophoresis.
After an overnight incubation in 5 mmol/L CaCl2 in TRIS
buffer, gels were stained with Coomassie R-250 in 10% acetic acid/40%
methanol and destained in 10% acetic acid/40% methanol. Clear areas
represent caseinolytic activity. Note caseinolytic bands at 50 to 55 kD corresponding in size to uPA, and that at 24 hours of hypoxic culture,
the caseinolytic bands were not discernible.
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Additional studies in which the levels of gelatinases in the
conditioned medium of HTR-8/SVneo cells cultured under standard or
hypoxic conditions were assessed by gelatin zymography showed that
hypoxia did not affect the amounts of these proteins released in
response to hypoxia (not shown). These observations suggest that the
increased invasiveness of HTR-8/SVneo cells observed under hypoxic
conditions is not attributable to the increased production or secretion
of these proteinases, and instead results from another mechanism,
potentially involving increased expression of uPAR.
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DISCUSSION |
Our results show that uPAR expression by human trophoblasts and
endothelial cells is regulated by hypoxia. Furthermore, they suggest
that these responses occur via a heme protein-dependent pathway. This
conclusion is based on the results of experiments in which the ability
of such a protein to regulate uPAR expression was assessed through the
use of cobalt chloride and Tiron, each of which causes the functional
displacement of ferrous iron from the heme moiety of the putative
oxygen sensing protein. We also showed that the effects of hypoxia on
uPAR expression were blocked by carbon monoxide. Although these results
are consistent with the hypothesis that the hypoxia-induced stimulation
of uPAR expression occurs through a heme protein, their absolute
dependence on such a protein remains unproven until it is identified
and cloned.
A similar pathway regulating the production of erythropoietin (EPO) has
been extensively characterized31 and shown to involve the
interactions of specific transcription factors, such as
HIF-1,32 with discrete enhancer regions in the
3-flanking sequences of the EPO gene.33
Despite restricted cellular production of EPO, hypoxic induction of
reporter genes containing the EPO enhancer has been observed in
a wide variety of cell types, suggesting that HIF-1 may also mediate
other adaptive responses to hypoxia.34 Inspection of the
5-flanking region of the uPAR gene35 shows three
potential HIF-1 binding sequences, each of which differs by only one
nucleotide from the consensus sequence 5-XACGTGCX-3, originally identified in the genes encoding EPO and several glycolytic enzymes.36 These sequences, 5-TTCGTGCT-3,
5-TAGGTGCT-3 and 5-TACGGGCC-3, are located
in the same orientation as the uPAR transcriptional unit,
between nucleotides -501 and -494, -578 and -573, and -1204 and -1197, respectively. We did not identify any potential HIF-1 binding sites in
the 3-flanking regions of the uPAR gene.37
In addition to HIF-1,33 hypoxia may induce the activation
of NF- B, c-fos, c-jun, c-jun B, and
jun D.38,39 While one group has also reported that
hypoxia induces the activation of c-Src,40 a recent
study failed to confirm these observations, despite culture of cells
under hypoxic conditions that led to the induction of HIF-1, EPO, VEGF,
and the glucose transporter-1, Glut-1.41 Furthermore,
perturbation of c-Src activity in Hep3B cells did not affect
the normoxic or hypoxic expression of the latter
proteins.41 Whether other factors, in addition to HIF-1, contribute to the hypoxia-mediated stimulation of uPAR expression is
unknown.
Although we propose that the increased expression of uPAR in response
to hypoxia occurs primarily through HIF-1-mediated stimulation of uPAR
gene expression, we also considered the possibility that these
observations may reflect an autocrine effect of endogenously-released growth factors in response to hypoxia. For example, VEGF, the expression of which is markedly increased in response to
hypoxia,1 stimulates the expression of uPAR by bovine and
human endothelial cells.25 In our studies, however, culture
of HTR-8/SVneo cells with exogenous VEGF did not result in increased
levels of uPAR mRNA. These results suggest that, at least under the
conditions used in these studies, the effect of hypoxia on uPAR
expression is not mediated through VEGF. This conclusion is supported
by the results of studies in which we observed that blocking anti-VEGF antibodies did not inhibit the increased expression of uPAR protein or
mRNA induced by hypoxia (T. Fitzpatrick, K.R. McCrae, and C.H. Graham,
unpublished observations, April 1997). However, it is possible that in other cell types, which produce greater amounts of
VEGF, increased expression of uPAR in response to hypoxia may occur
through additional pathways.
We observed that the levels of urokinase were progressively reduced in
the conditioned medium of HTR-8/SVneo cells cultured for increasing
intervals under hypoxic conditions. Although we have not determined
precisely the mechanisms accountable for this observation, we believe
it is likely that these decreased levels reflect increased binding of
secreted uPA to cellular uPAR. This hypothesis is supported by the
observation that cells cultured under hypoxic conditions expressed
increased cell surface plasminogen activator activity. Nevertheless, we
believe that the increased expression of uPAR induced by hypoxia is
likely to be of importance in cellular migration and invasion in vivo,
as it has been shown, for example, that invasive neoplastic cells may
use uPA produced by neighboring stromal cells to facilitate invasion
through extracellular barriers.42 Similar considerations
apply to the increased kd for binding of uPA to cellular uPAR after
exposure of cells to hypoxia. This modest increase, which is similar to
that which accompanies the increased expression of uPAR in response to
phorbol 12-myristate, 13-acetate (PMA),43 appears to result
from additional glycosylation or other posttranslational modifications
of the receptor. However, an increase in the kd of the magnitude
observed here is not likely to significantly affect the degree of uPAR saturation with uPA in the presence of either increased endogenous uPA
production or the production of significant amounts of uPA by
neighboring cells. Finally, even in our system, the net effect of
exposure of HTR-8/SVneo cells to hypoxia was an increase in the
expression of cell surface PA activity, which we believe underlies the
increased cellular invasiveness that occurred in parallel. Whether the
increased in vitro invasiveness and in vivo metastatic capability of
murine sarcoma cells under hypoxic conditions involve similar
mechanisms has not yet been determined.5
Our findings may be relevant to several biological processes in which
cell migration and invasion occur. For example, increased expression of
uPAR by extravillous trophoblast cells may facilitate their invasion
through hypoxic regions of the superficial endometrial stroma.44 In support of this hypothesis are studies in
which trophoblast invasion of the uterine wall has been shown to be increased in pregnant rhesus monkeys in which uteroplacental blood flow
was impaired following constriction of the abdominal
aorta.45 It should also be noted that the human placenta is
relatively hypoxic during the first 10 weeks of gestation,8
a period when trophoblast cells are maximally invasive. In addition,
increased expression of uPAR is enhanced in migrating endothelial cells in vitro,46 and the fact that neovascularization of
transplanted tumors in vivo is inhibited by uPAR
antagonists47,48 suggests a role for hypoxia-stimulated
uPAR expression in tumor angiogenesis. Additional studies have shown
that uPAR antagonists inhibit tumor metastasis,49,50 as
well as local invasion,50,51 perhaps explaining, in part,
the association of elevated uPAR levels in extracts of breast and other
types of carcinomas with a poor clinical outcome.51
Finally, the expression of hypoxia-inducible proteins by macrophages
within atherosclerotic plaques shows that hypoxia occurs within this
setting as well,6 and suggests that the migration of these
and other cell types within the plaque may be facilitated by
hypoxia-stimulated increases in uPAR.52
In conclusion, we have shown that the expression of uPAR is stimulated
by hypoxia and that this response may involve an oxygen-sensing heme
protein. Hence, we believe that the uPAR should be added to the
expanding list of hypoxia-inducible proteins induced through such a
pathway. Although it is likely that HIF-1 is involved in the
hypoxia-mediated increase in uPAR expression, additional studies will
be required to address this issue in different cell types and determine
the physiologic importance of these observations.
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FOOTNOTES |
Submitted September 30, 1997;
accepted December 19, 1997.
Supported by Grant No. T-3361 from the Heart and Stroke Foundation of
Ontario (to C.H.G.), Grant No. HL 50827 from the National Institutes of
Health, and Grant No. 95-0220 from the American Heart Association (to
K.R.M.). C.H.G. is a Research Scholar from the Heart and Stroke
Foundation of Canada.
Address reprint requests to Charles H. Graham, PhD,
Department of Anatomy and Cell Biology, Botterell Hall, 9th Floor,
Queen's University, Kingston, Ontario, Canada K7L 3N6.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful for the technical assistance of Shannyn
Macdonald-Goodfellow and for the photographic work of Bob Temkin. We
also thank Dr Jim Johnson for providing assistance with the flow
cytometric analysis.
| |
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Abstract
Introduction
Abstract