From the Division of Developmental and Newborn
Biology and ¶ Division of Newborn Medicine, Department of
Medicine, Children's Hospital, Harvard Medical School, Boston,
Massachusetts 02115 and the
Center for Medical Genetics,
Departments of Pediatrics and Medicine, The Johns Hopkins University
School of Medicine, Baltimore, Maryland 21287
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ABSTRACT |
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Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and blood vessel remodeling. Its expression is up-regulated in vascular smooth muscle cells by a number of conditions, including hypoxia. Hypoxia increases the transcriptional rate of VEGF via a 28-base pair enhancer located in the 5'-upstream region of the gene. The gas molecules nitric oxide (NO) and carbon monoxide (CO) are important vasodilating agents. We report here that these biological molecules can suppress the hypoxia-induced production of VEGF mRNA and protein in smooth muscle cells. In transient expression studies, both NO and CO inhibited the ability of the hypoxic enhancer we have previously identified to activate gene transcription. Furthermore, electrophoretic mobility shift assays indicated decreased binding of hypoxia-inducible factor 1 (HIF-1) to this enhancer by nuclear proteins isolated from CO-treated cells, although HIF-1 protein levels were unaffected by CO. Given that both CO and NO activate guanylyl cyclase to produce cGMP and that a cGMP analog (8-Br-cGMP) showed a similar suppressive effect on the hypoxic induction of the VEGF enhancer, we speculate that the suppression of VEGF by these two gas molecules occurs via a cyclic GMP-mediated pathway.
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INTRODUCTION |
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Low oxygen tension is a potent regulator of diverse biological processes, including erythropoiesis, angiogenesis, and vascular cell contractility. These effects are mediated by several proteins that are induced under hypoxic environments and modulate cell-cell interactions, cell proliferation, and differentiation. In the vasculature, hypoxia regulates the expression of genes encoding growth factors such as endothelin-1 (ET-1)1, platelet-derived growth factor-B (PDGF-B) and vascular endothelial growth factor (VEGF), as well as genes regulating the production of gas molecules such as nitric oxide (NO) and carbon monoxide (CO) (1-5). Whereas the expression of the endothelial nitric oxide synthase gene is suppressed by hypoxia, the expression of heme oxygenase-1 (HO-1), the enzyme catalyzing the production of CO, is up-regulated by hypoxia (5).
Mechanisms by which hypoxia alters gene expression include transcriptional and post-transcriptional regulation (4, 6, 7). Several hypoxia-responsive cis-acting elements have been identified (8, 9). We have reported the presence of a 28-bp enhancer located approximately 980 bp upstream of the VEGF transcription start site, which is necessary and sufficient to up-regulate transcription of the VEGF gene in response to hypoxia (10). This hypoxia response element contains a sequence homologous to (and now has been included into) the hypoxia-inducible factor 1 (HIF-1) consensus (11). HIF-1 is a basic helix-loop-helix transcription factor originally identified to mediate the transcriptional activation of the erythropoietin gene (8) leading to enhanced erythropoiesis under hypoxia. It was subsequently shown to regulate the expression of genes encoding glycolytic enzymes (12) and the gene for VEGF (10, 11) implicating it as an important regulator of the cellular responses to hypoxia. HIF-1 itself is also regulated by hypoxia at the posttranscriptional level (13-15), but intracellular events regulating its DNA binding activity and function are poorly understood.
Gas molecules are not only regulated by hypoxia but can also modify the effects of hypoxia on other genes. We reported that endothelial-derived NO inhibits both the basal and hypoxia-induced expression of ET-1 and PDGF-B genes in endothelial cells (16). Smooth muscle cell-derived CO was also shown to inhibit the hypoxic induction of these genes in a paracrine manner (17). In this report we investigated mechanisms by which NO and CO modulate hypoxic signal transduction leading to altered gene expression. We demonstrate that NO and CO inhibited the hypoxic induction of VEGF at the transcriptional level. This was due to decreased HIF-1 DNA binding activity, although HIF-1 protein levels actually increased. The uncoupling of HIF-1 production and HIF-1 function by NO and CO suggests the existence of additional control points for hypoxic signal transduction lying downstream of the putative oxygen sensor.
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EXPERIMENTAL PROCEDURES |
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Media and Cell Culture-- Bovine pulmonary artery endothelial cells (BPAEC), rat aortic smooth muscle cells (RASMC), and fetal smooth muscle cells (A7r5) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 0.1 mg/ml gentamicin sulfate. The cells were exposed to a normoxic environment consisting of 21% O2, 5% CO2, balance N2, or to a hypoxic mixture of 95% N2 and 5% CO2. The measured PO2 in the medium was 18-20 mm Hg under hypoxic conditions, as we reported previously (2). Cells were cultured at 37 °C in humidified incubators.
Northern Blot and RNA Analysis--
Total cellular RNA was
isolated from cultured RASMC by guanidinium isothiocyanate extraction
method. Total RNA (15 µg) was electrophoresed in 1% agarose gels
containing formaldehyde and transferred to nitrocellulose membranes by
blotting. The filters were hybridized with a cDNA probe specific
for the rat VEGF gene (18). A mouse -actin probe was used to
normalize for RNA loaded. The cDNA fragments were labeled with
[
-32P]dCTP using a standard random-primed reaction to
a specific activity of 1-2 × 109 cpm/µg. The
membranes were hybridized for 2 h at 68 °C in QuikHyb solution
(Stratagene, La Jolla, CA) with 2 × 106 cpm/ml of
probe and washed twice in 2 × SSC containing 0.1% SDS at
60 °C for 30 min and were then exposed to film (X-Omat AR; Eastman
Kodak Co.) with intensifying screens at
80 °C. For quantitation, we scanned autoradiographs with a laser densitometer (Ultroscan XL; LKB
Instruments, Inc., Bromma, Sweden) running the Gel Scan XL software
package (Amersham Pharmacia Biotech).
Transfection and Reporter Gene Assays--
Transfections of
BPAEC were carried out on cells at 50-80% confluence using
LipofectAMINE reagent (Life Technologies, Inc.) according to the
manufacturer's protocol. RASMC were transfected using DEAE-dextran
method (19). Cell lysis was performed using the reporter gene lysis
buffer from Promega (Madison, WI), and the activities of
-galactosidase and CAT were measured according to product diffusion
method (20). Normalized CAT activity was the ratio of radioactivity (in
counts/min) of labeled acetylchloramphenicol to the optical density
units from the cleavage product of
o-nitrophenyl-
-D-galactopyranoside catalyzed
by
-galactosidase.
Nuclear Protein Extraction and Electromobility Shift Assay-- Nuclear proteins were isolated from BPAEC using the method described by Schreiber et al. (21), and the total protein was quantified with the Bio-Rad protein assay. Electromobility shift assay was performed according to the protocol described previously using as a probe a 35-bp DNA fragment (A-G) that contains the 28-bp hypoxic enhancer (10).
VEGF Peptide Quantification--
RASMC were cultured in 100-mm
plates in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum as above. When the cells were approximately 80%
confluent, the medium were changed to Dulbecco's modified Eagle's
medium with 1% fetal bovine serum, and the cells were exposed to
normoxic or hypoxic conditions for 48 h as above. The culture
medium from these confluent cells were collected and concentrated
30-fold using Centriprep-10 (Amicon, Inc., Danvers, MA). They were
subsequently analyzed using a commercially available sandwich enzyme
immunoassay for VEGF165 peptide (QuantikineTM,
R & D Systems, Minneapolis, MN). Values were expressed in
picograms/ml, and the sensitivity of the assay was 5.0 pg/ml. The cells
were trypsinized and counted in a Coulter counter, and the
concentration of VEGF was reported in picograms/106 cells
to normalize for cell number. To assess the effect of endogenously produced CO on the production of VEGF under hypoxia, the same experiment was done after addition of tin protoporphyrin IX (SnPP-9) or
hemoglobin (Hb) to the medium at a final concentration of 100 and 50 µM, respectively, followed by hypoxic exposure for
48 h. Hb was purchased from Sigma and was prepared by treatment
with excess reducing agent, sodium hydrosulfite (22). The experiment was performed five times in duplicate and the mean VEGF concentrations were compared using the Kruskal Wallis nonparametric analysis of
variance test with a level of significance of p 0.05.
Western Analyses--
Nuclear proteins were extracted from BPAEC
according to the method described previously (23). A total of 20 µg
of nuclear proteins isolated from cells treated with different
conditions was loaded on a 7% SDS-polyacrylamide gel. HIF-1 and
HIF-1
were detected by the ECL Western kit from Amersham Pharmacia
Biotech. Affinity-purified antibodies against HIF-1
and HIF-1
(24) were used at 1:1000 dilution.
Reagents-- SnPP-9 was purchased from Porphyrin Products, Inc. (Logan, UT). All other reagents used were obtained from Sigma unless otherwise indicated.
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RESULTS |
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CO and NO Suppress the Hypoxic Induction of VEGF mRNA Expression-- RASMC were exposed to hypoxia or normoxia for 3-48 h, and total cellular RNA was isolated for Northern analysis. After hybridization with the rat VEGF probe a 4-fold increase in VEGF mRNA was detected in cells exposed to hypoxia for 12 h, compared with the normoxic controls (Fig. 1A). Levels of VEGF mRNA started to increase at 6 h of hypoxia, reached a maximum at 12-24 h, and declined by 48 h. To examine the effect of NO on VEGF mRNA, cells were treated with the NO donor, S-nitrosoglutathione (GSNO), for 12 h under hypoxic or normoxic conditions. GSNO (50-500 µM) reduced VEGF mRNA levels under hypoxia dose-dependently (Fig. 1B, lanes 4 and 6, respectively). Similar results were obtained with treatment of cells with 500 µM sodium nitroprusside, another NO donor (data not shown).
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CO Regulates the Levels of Secreted VEGF Protein by Smooth Muscle Cells-- To verify that the VEGF mRNA levels changed in parallel with levels of VEGF peptide secreted into the medium, we measured VEGF concentrations in the RASMC-conditioned medium. Fig. 2 shows the VEGF concentrations in the medium under various experimental conditions. Using an enzyme-linked immunosorbent assay, we were unable to detect any VEGF peptide in quiescent normoxic smooth muscle cells and detected only very low levels of protein in the conditioned medium of cells after 48 h of hypoxic exposure (mean = 0.2 pg/106 cells). However, VEGF protein was consistently detected in the conditioned media after 48 h of hypoxic exposure in the presence of either SnPP-9 or Hb. The mean VEGF protein concentration after 48 h of hypoxic exposure in the presence of SnPP-9 or Hb was 0.61 pg/106 cells. This was significantly higher than normoxia and 3-fold higher than hypoxia alone (p < 0.01). Although SnPP-9 is an inhibitor for both nitric oxide synthases and heme oxygenases and hemoglobin can scavenge both CO and NO, transcripts of NO synthase (inducible or constitutive) have not been detected in RASMC at any O2 concentration, and inhibitors of NO synthesis had no effect on smooth muscle cell-derived cGMP levels (5). This suggests that in cultured RASMC, endogenous CO is the predominant negative regulator of VEGF production under hypoxic conditions.
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CO and NO Suppress the Hypoxic Induction of VEGF through
Attenuation of the Hypoxic Enhancer--
Previous work has shown that
hypoxia induces VEGF gene transcription through the activation of its
enhancer (10, 25, 26), a 28-bp region that contains a HIF-1 consensus.
To investigate whether the inhibitory effects of CO and NO occur at the
transcriptional level, we tested the effect of these two gases on the
expression of a reporter gene under the control of the VEGF hypoxic
enhancer. Cultured BPAEC were transfected with reporter plasmid
pV111/CATa, which contains the hypoxic enhancer upstream of the
thymidine kinase promoter fused to the CAT gene (10), and GSNO was
added to the medium at a concentration of 0.5 mM. After
incubation for 24 h, cells were lysed and the reporter gene (CAT)
activity was measured and normalized to -galactosidase. We observed
that in hypoxic cells, 0.5 mM GSNO reduced CAT activity by
50%. In normoxic cells the relative CAT activity was slightly
increased by treatment with GSNO. The combined results from these
effects are that the hypoxic induction was reduced from 18.4- to
4.6-fold by GSNO (Fig. 3A). A
higher concentration of GSNO (5 mM) led to cell death and resulted in undetectable reporter gene activity (data not shown). In
RASMC, GSNO inhibited the hypoxic induction in a
dose-dependent manner. At 1 mM of GSNO, the
hypoxic induction was 8.7-fold (Fig. 3B, column
1), which is comparable with untreated hypoxic cells (data not
shown). At 2 mM, the induction was reduced to 1.8-fold (column 2), and at 3 mM, the hypoxic induction
was completely abolished (column 3).
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CO Regulates the Binding of Hypoxia-inducible Factor 1 to the VEGF
Enhancer--
HIF-1 can specifically bind to the VEGF enhancer (10,
11). In addition to the HIF-1 binding site, there is a downstream sequence that is also required for function (10). We analyzed expression of the HIF-1 and HIF-1
subunits and HIF-1 DNA binding activity to the 28-bp hypoxia response element to determine whether CO
affects the production of HIF-1 and/or its binding to the enhancer. Expression of HIF-1
protein in BPAEC was dramatically induced by
12 h of hypoxia, and the induction was not markedly affected by CO
levels (Fig. 4A). HIF-1
protein was also increased by hypoxia, but to a much lesser extent, and
was also unaffected by exogenous CO (Fig. 4B). However, the
binding of HIF-1 to the enhancer was greatly reduced compared with the
binding observed in cells treated with hypoxia alone (Fig.
4C, arrow). The lower band, which represents the
constitutive binding, is not changed by CO treatment, indicating that
CO exerts its inhibitory effect by specifically regulating the binding
activity of HIF-1.
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cGMP Levels Regulate VEGF Enhancer Function-- Both NO and CO stimulate the activity of guanylyl cyclase (27, 28), which catalyzes the production of cGMP, an important second messenger for signal transduction. To determine whether the suppressant effect of CO and NO on the hypoxic induction of VEGF enhancer was through the action of cGMP, a cGMP analog, 8-Br-cGMP, was added to the culture medium to the concentration of 1 mM, and reporter gene activity was assayed using RASMC exposed to hypoxia or normoxia. In the presence of 8-Br-cGMP, the hypoxic induction of VEGF enhancer was suppressed from 8.9- to 4.9-fold (Fig. 5), paralleling the effect of dibutyryl cGMP on endogenous VEGF gene expression (Fig. 1C) and confirming the involvement of cGMP in this process.
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DISCUSSION |
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VEGF plays an important role in the process of angiogenesis and
vascular remodeling (29). Its expression has been shown to be regulated
by various agents, such as transforming growth factor- (30),
estrogen (31), prostaglandin E2 and E1 (32), interleukin-1
(33), as well as hypoxia (25, 34, 35). Our previous
study identified an enhancer in the human VEGF promoter region, which
is necessary and sufficient to provide hypoxic induction when tested in
a reporter system (10). The data in this report demonstrate that
induction of VEGF gene transcription via this hypoxic enhancer is
subject to modulation by NO and CO, two gas molecules that are
important in the regulation of blood vessel tone (36, 37).
NO has been reported previously to activate or suppress gene
expression. For example, NO can activate gene transcription from AP-1-responsive promoters in mammalian cells (38), an effect mimicked
by 8-Br-cGMP, indicating that NO may act by stimulating guanylyl
cyclase production of cGMP. In contrast, NO suppresses the mRNA
levels of macrophage-colony-stimulating factor induced by oxidized low
density lipoprotein or tumor necrosis factor- (39). This suppression
was associated with decreased DNA-binding activity of the transcription
factor NF-
B but was not mediated by cGMP. We have reported
previously that NO can suppress the hypoxic induction of both ET-1 and
PDGF-B gene expression (16) in vascular endothelial cells. Using the
VEGF gene as a model, we once again demonstrated a suppressive effect
of NO on the hypoxic induction of genes expressed by vascular cells. In
this study, NO donors were able to suppress both the transcription of a
reporter gene containing the VEGF hypoxic enhancer and the binding of
the transcriptional activator HIF-1 to the enhancer. In contrast to the
NO effect on the induction of macrophage-colony-stimulating factor,
cGMP could mimic the suppressive effect of NO.
Earlier studies have shown that NO plays a role in microvascular
permeability. An analog of L-arginine,
N-nitro-L-arginine methyl
ester, which can compete with L-arginine and suppress the
activity of NO synthase, caused a rapid increase in microvascular
permeability (40). Furthermore, the cytosolic second messenger cGMP has
been shown to be the mediator between NO and the changes observed in
vascular permeability (41). The mechanism by which cGMP regulates
microvascular permeability is not clearly understood. It has been
suggested that intracellular cGMP activates cGMP-dependent
protein kinases that affect myosin phosphorylation. The modifications
on the cytoskeletal filaments induce endothelial cell relaxation or
contraction and change the size of interendothelial cell junctions,
leading to the altered vascular permeability (41). In our study, we
found that NO and cGMP suppressed the transcriptional rate of the VEGF
gene. Since VEGF is a well known endothelial cell-specific vascular
permeability factor (42-44), our results provide an alternative
explanation for the NO effect on vascular permeability. NO can regulate
levels of cGMP, which in turn modulate the expression of VEGF in
vascular smooth muscle cells and in endothelial cells. The changed VEGF production and secretion from smooth muscle cells or endothelial cells
can alter the permeability of vascular endothelium in a paracrine or an
autocrine fashion, respectively. It should be noted that our results
are not in contradiction to the possible pathway involving a
cGMP-dependent protein kinase. In fact, protein phosphorylation events also occur following VEGF stimulation of endothelial cells. Thus, NO effects on an NO-dependent
protein kinase and on VEGF expression could co-exist and be part of a complex system that regulates vascular permeability.
Given the similarities between NO and CO, one would expect that CO will have a similar physiological effect as NO. Indeed, previous studies by our group have shown that CO is produced in vascular smooth muscle cells and regulates cGMP levels in these cells (5). Furthermore, we and others have demonstrated the inhibitory effect of CO on the hypoxic induction of certain genes, including ET-1, PDGF (17), and VEGF (7). However, the molecular mechanisms of this regulation have not been reported previously. From the results reported here, we demonstrate that CO can regulate the transcriptional rate of a target gene and that for VEGF this effect occurs at the level of the hypoxic enhancer. Furthermore, we have shown that the binding of HIF-1 to the enhancer element was reduced by CO treatment and that this effect was "mimicked" by cGMP. This interference with the binding activity of HIF-1 suggests potential changes in phosphorylation or possibly CO/NO-mediated redox effects on HIF-1 dimerization and/or DNA binding. Wang and Semenza (45) have reported that phosphorylation of HIF-1 is required for DNA binding, and other reports (46, 47) have shown that HIF-1 protein is stabilized by a redox-dependent signaling pathway.
Our findings that CO inhibits HIF-1 DNA binding activity under hypoxia
without altering the hypoxia-induced increases in HIF-1 protein may
indicate that the molecular mechanisms of CO modulation of hypoxic
signal transduction do not involve a putative heme protein
O2 sensor at the apex of the cascade (48). They suggest the
existence of additional control points, possibly intracellular protein
modification events, that are downstream of O2 sensing and
upstream of HIF-1 action. The results presented in this paper provide further evidence for the essential role of HIF-1 in the regulation of VEGF transcription in hypoxic cells. Posttranscriptional events also regulate the steady state levels of VEGF mRNA (25, 26).
However, whether CO can effect these processes remains to be
investigated.
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ACKNOWLEDGEMENT |
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We thank Amy Elias for her expert assistance in preparation of this manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by an Individual National Research Service Award HL-09008.
** Supported by the American Heart Association and by National Institutes of Health Grants R01 DK39869 and R01 HL55338. Established Investigator of the American Heart Association.
Supported by the American Heart Association and by National
Institutes of Health Grants SCOR 1P50 HL56398 and R01 HL55454. To whom
correspondence should be addressed: Children's Hospital, 300 Longwood
Ave., Enders 9, Boston, MA 02115.
1 The abbreviations used are: ET-1, endothelin-1; PDGF-B, platelet-derived growth factor-B; VEGF, vascular endothelial growth factor; NO, nitric oxide; CO, carbon monoxide; HO-1, heme oxygenase-1; HIF-1, hypoxia-inducible factor 1; BPAEC, bovine pulmonary artery endothelial cells; RASMC, rat aortic smooth muscle cells; SnPP-9, tin protoporphyrin IX; Hb, hemoglobin; GSNO, S-nitrosoglutathione; bp, base pair(s); CAT, chloramphenicol acetyltransferase.
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REFERENCES |
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