© 1995 American Heart Association, Inc.
Articles |
Molecular Regulation of the Bovine Endothelial Cell Nitric Oxide Synthase by Transforming Growth Factor–ß1
From the Departments of Medicine and Pharmacology, Emory University School of Medicine, and the Atlanta Veterans Administration Medical Center, Atlanta, Ga.
Correspondence to David G. Harrison, MD, Cardiology Division, PO Box LL, Emory University School of Medicine, Atlanta, GA 30322.
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Abstract The promoter region of the endothelial cell nitric oxide synthase (ecNOS) gene contains potential response elements for transforming growth factor–ß1 (TGFß1). TGFß1 plays an important role in the pathogenesis of atherosclerosis, vascular hypertrophy, and angiogenesis. We therefore sought to determine whether TGFß1 might modulate ecNOS expression in bovine aortic endothelial cells (BAEC). TGFß1 increased ecNOS mRNA in a dose-dependent manner. TGFß1 also increased ecNOS protein content. The production of nitrogen oxides (NOx), assessed by chemiluminescence, and nitric oxide synthase activity, assessed by arginine/citrulline conversion, were increased in TGFß1-treated cells. Transcriptional activity of the 5'-flanking promoter region of the ecNOS gene was increased by TGFß1, as assessed by transfection with promoter/luciferase constructs. Deletion analysis suggested that the TGFß1-response element was present between nucleotides -1269 and -935 from the first transcription start site, in which a putative nuclear factor–1 (NF-1) binding site existed. Gel shift assays showed that nuclear protein(s), immunologically similar to CCAAT transcription factor/NF-1, bound to the putative NF-1 binding site in a sequence-specific manner. Mutation of the putative NF-1 binding site in the promoter/luciferase construct significantly decreased the responsiveness to TGFß1. In conclusion, TGFß1 increases ecNOS expression associated with an increase in production of NO in BAEC. This response is probably mediated by transcriptional activation of the ecNOS gene promoter.
Key Words: nitric oxide synthase • transforming growth factor–ß1 • endothelium • nuclear factor–1
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Introduction |
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The endothelium regulates vascular tone by producing prostacyclin, EDRF, and endothelium-derived constricting factors. The EDRF is a labile potent vasodilator, which is NO or a related compound.1 2 Recently, ecNOS cDNA3 4 5 6 has been cloned and the 5'-flanking region of the gene sequenced.7 8 9 A variety of cis-regulatory sequences, such as potential AP-1 and AP-2 binding sites, sterol-regulatory elements, shear stress response elements, and a putative NF-1 binding site are present in the promoter region of the ecNOS gene.7 8 Although the ecNOS is considered a constitutive enzyme, recent evidence suggests that chronic shear stress, tumor necrosis factor, and proliferating state influence the level of ecNOS mRNA. Thus, various physical and humoral stimuli may regulate its expression.3 10 11
TGFß1, a 25-kD homodimeric multifunctional
peptide, plays an important role in the pathogenesis of
atherosclerosis, vascular hypertrophy in hypertension, vascular
response to injury after balloon angioplasty, and angiogenesis in wound
healing.12 13 14 15 TGFß1 modulates a wide variety
of cellular activities through transcription of various genes. The
precise molecular mechanism of gene expression induced by
TGFß1 is still unknown, although several mechanisms have
been proposed. The TGFß1-activating element of the
promoter region of the mouse 
2(I) collagen gene is very similar to a
consensus sequence NF-1 binding site [TGG(N)6GCCAA], and
NF-1 mediates expression of this gene.16 NF-1 is also
reported to mediate the increases in PAI-1 gene expression induced by
TGFß1.17 In contrast, TGFß1
autoinduction is mediated by a signaling pathway involving AP-1
regulatory elements.18 Both putative NF-1 and AP-1 binding
sites are present in the 5'-flanking promoter region of the bovine
ecNOS, -1014 and -441 nucleotides from the first transcription
start site, respectively.8 On the basis of these
considerations, we examined the effect of TGFß1 on gene
expression and protein induction of ecNOS, NO production, and the
activity of NOS in cultured BAECs.
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Methods |
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Cell Culture
BAECs were grown in culture as previously described.19 The cells were cultured in Medium 199 supplemented with 10% fetal calf serum (Hyclone Laboratories). The cells used in the present study were from the fifth to the ninth passage, and were studied upon reaching confluency.
Northern Blot Analysis
Northern blot analysis was performed as previously
described.3 11 Total RNA was isolated by phenol
extraction, size-fractionated on a 1% agarose/3% formaldehyde gel,
and transferred to a nitrocellulose membrane. Hybridizations were
performed overnight at 42°C with a [32P]dCTP-labeled
ecNOS cDNA fragment obtained by Sst I (GIBCO BRL) digestion
corresponding to sequences from nucleotides 941 to 2099 of
the full-length ecNOS cDNA. The membranes were then washed twice with
2x standard saline citrate and 1% SDS for 30 minutes at 55°C and
subsequently once with 0.2x standard saline citrate and 0.1% SDS for
30 minutes at 55°C.
In all studies, the nitrocellulose membranes were stripped and subsequently hybridized with cDNA for GAPDH to serve as a control. The autoradiographs were quantified by densitometric scanning.
Western Blot Analysis
As previously described,3 11 Western blot
analysis was performed with a rabbit antibody against BAEC NOS
(antibody p459) and a goat anti-rabbit secondary antibody conjugated to
horseradish peroxidase. The polyclonal antibody p459 was raised against
the peptide sequence 628 to 638 of the bovine ecNOS linked to BSA and
then immunopurified. This peptide sequence does not exist in the
macrophage enzyme, and in preliminary studies p459 did not
detect macrophage NOS by either Western blot analysis
or immunohistochemistry. Signals were detected with the ECL detection
system (Amersham Corp) on a standard x-ray system.
Measurement of Endothelial Cell NO
Production
The production of NO from BAECs was assessed by
measurement of nitrite and nitrate, the stable degradation products
of NO, with a chemiluminescence technique, as previously
described.20 The amounts of NO released were normalized to
the number of cells in each culture dish.
Measurement of NOS Activity
The activity of NOS was assessed by conversion of
[3H]arginine to [3H]citrulline, as
previously described.11 Cell homogenates were
incubated in the reaction buffer ([mmol/L] Tris HCl 50, EDTA 0.1,
EGTA 0.1, pH 7.5) containing calmodulin 100 nmol/L,
CaCl2 2.5 mmol/L, NADPH 1 mmol/L, tetrahydrobiopterin 3
µmol/L, and the substrate L-arginine 100 µmol/L
combined with L-[2,3-3H]arginine (0.2
µCi; specific activity, 55 Ci/mmol) for 15 minutes at 37°C. After
incubation, the reaction was quenched by the addition of 1 mL of the
stop buffer ([mmol/L] EDTA 2, EGTA 2, HEPES 20, pH 5.5). The reaction
mix was applied to a 1-mL column containing Dowex AG 50WX-8
(Na+ form) resin (Bio-Rad).
L-[2,3-3H]Citrulline was eluted twice with
0.5 mL of the stop buffer and radioactivity was determined by liquid
scintillation counting.
Transfection of BAECs With Promoter/Reporter
Constructs
To determine whether the TGFß1 effect on ecNOS
gene expression was mediated by the 5'-flanking region of the gene, a
series of bovine ecNOS promoter/luciferase reporter gene plasmids were
constructed as recently described.8 Deletion fragments of
the bovine endothelial gene promoter with 5' end points
at nucleotides -2835, -1548, -1269, -935, -614, and
-416 and a 3' end point at nucleotide +240 were created by
PCR amplification of bovine genomic DNA. Fragments were then subcloned
into the promoterless plasmid, poLuc, upstream from a luciferase
reporter gene. Subconfluent BAECs were transfected with
promoter/luciferase constructs, a poLuc (negative control) construct,
or a pSV2Luc (positive control) construct containing the SV40
promoter/enhancer. BAECs were cotransfected with a pSV2CAT construct
containing the CAT gene to normalize for variation in transfection
efficiencies. Luciferase and CAT activities were determined 48 hours
after transfection.8
Gel Mobility Shift Assay
Nuclear extracts from BAECs were prepared as previously
described.21 Cell pellets were suspended in buffer A
([mmol/L] KCl 10, MgCl2 1.5, dithiothreitol 1, HEPES 10)
containing the proteinase inhibitors PMSF 1 mmol/L,
leupeptin 10 µg/mL, antipain 10 µg/mL, and aprotinin 0.023 trypsin
inhibitor units/mL. After centrifugation, the cell
pellets were resuspended in buffer A with 0.1% Triton X-100. After
incubation for 10 minutes at 4°C, the mixture was centrifuged
at 4°C for 5 minutes at 750g to obtain nuclear pellets.
The nuclear pellets were resuspended in the extraction buffer
([mmol/L] NaCl 420, MgCl2 1.5, EDTA 0.2, dithiothreitol
1, and HEPES 20 and glycerol 25%, pH 7.5) containing the same
proteinase inhibitors. After incubation for 30 minutes at
4°C, nuclear extracts were obtained by centrifugation
at 20 000g for 10 minutes. Synthesized double-stranded
oligonucleotides were labeled with
[
-32P]ATP at the 5' end with T4
polynucleotide kinase (Promega Corp). The nuclear extracts
were then incubated with approximately 100 000 cpm of radiolabeled
double-stranded oligonucleotides for 30 minutes at
30°C in 20 µL of binding solution ([mmol/L] KCl 150,
MgCl2 5, EDTA 1, dithiothreitol 1, PMSF 1, HEPES 12, and
Tris HCl 4 and glycerol 12%, pH 7.9) in the presence of 2 µg poly
dIdC and 4.5 µg BSA. In some experiments, before radiolabeled probes
were added, cold competitors were preincubated with nuclear extracts
for 15 minutes at room temperature. In other studies, a rabbit
polyclonal antibody directed against CTF/NF-1 (generously provided by
Dr Naoko Tanese and Dr Robert Tjian) or preimmune serum was
preincubated with nuclear extracts for 45 minutes at 37°C. The
incubated mixtures were separated on 4% polyacrylamide gels.
The gels were dried under vacuum and exposed to x-ray film. Sequences
of the oligonucleotides were as follows: ecNOS promoter
containing a putative NF-1 binding site (corresponding to sequences
from nucleotides -1006 to -1034 in the bovine ecNOS
promoter), 5'-CACCTTCTTGGTGTGACCCCAGAACTCGC-3';
random oligonucleotide,
5'-GCTGACTATGACAGCTTCGGAACTTGCTAC-3'; consensus NF-1
oligonucleotide,
5'-CCTTTGGCATGCTGCCAATATG-3' (Promega Corp);
and NF-1 mutant NOS promoter fragment,
5'-CACCTTCTGTTTGTGA-CCAACGAACTCGC-3'.
Complementary sequences were used to produce double-stranded DNA.
Site-Directed Mutagenesis of NF-1 Binding Site in ecNOS
Promoter/Reporter Construct
A 6-bp substitution of the putative NF-1 binding site was
introduced into the ecNOS -1548/+240/luciferase construct by the
overlapping extension PCR technique.22 A synthetic
oligonucleotide (corresponding to the sequence from
nucleotides -1049 to -990 in the bovine ecNOS promoter)
containing a 6-bp substitution of the NF-1 binding site was used as a
mutagenic primer and the ecNOS -1548/+240/luciferase construct was
used as a template. This mutated oligonucleotide
produced by PCR was subcloned into the ecNOS -1548/+240/luciferase
construct in the Tth111 and BamHI sites. By use
of this approach the putative NF-1 binding site
(5'-TGGTGTGACCCCA-3') was mutated to
5'-GTTTGTGACCAAC-3'.
Incorporation of the mutation was confirmed by dideoxy
chain termination sequencing with Sequenase 2.0 (United States
Biochemical).
DNA Synthesis and Cell Growth Study
DNA synthesis was assessed by measurement of the incorporation
of [methyl-3H]thymidine into the acid-insoluble material
as previously described.23 To achieve quiescence, BAECs on
12-well dishes were cultured in Medium 199 with 0.1% BSA for 36 hours.
Quiescent cells were cultured for 24 hours in Medium 199 with 5% fetal
calf serum containing or lacking TGFß1. Finally, the
cells were incubated for 24 hours in the same medium supplemented with
2 µCi [methyl-3H]thymidine. After precipitation with
2% trichloroacetic acid, incorporated radioactivity was measured by
liquid scintillation counting. In separate experiments, for examination
of the effect on cell growth, cell numbers were determined by counting
with a hemacytometer.
Materials
TGFß1 was used as the homodimer isolated from
porcine platelets (R&D Systems). Radiochemicals were purchased from
Amersham Corp. All other reagents were purchased from Sigma Chemical Co
except where specified.
Statistical Analysis
The data in this article are expressed as mean±SD. Comparisons
of data between two groups were made by unpaired Student's
t test. Values of P<.05 were considered
significant.
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Results |
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TGFß1 Increased mRNA of ecNOS in BAECs
Levels of ecNOS transcripts were increased as early as 3 hours after exposure to 1 ng/mL TGFß1 (Fig 1A
).
Maximal induction, observed at 12 hours, was by approximately 2.3-fold
(three different experiments). The level of mRNA began to decrease at
24 hours.
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TGFß1 (0.01 to 10 ng/mL for 6 hours) increased in ecNOS
transcripts in a dose-dependent manner (Fig 1B
). The effect was
detectable in the presence of 0.01 ng/mL TGFß1.
To determine whether the increase in ecNOS mRNA was caused by
enhanced transcription, BAECs were exposed to 10 µg/mL actinomycin D
for 30 minutes before and during exposure to 1 ng/mL
TGFß1 for 6 hours. Actinomycin D slightly decreased the
level of ecNOS mRNA in control cells, whereas it abolished the increase
in ecNOS mRNA in TGFß1-treated cells (Fig 1C).
TGFß1 Increased ecNOS Protein Expression in
BAECs
Stimulation of BAECs with 1 ng/mL of TGFß1 for 24
hours potentiated ecNOS protein expression 2.0-fold (in three different
experiments assessed by densitometry) (Fig 2A).
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TGFß1 Potentiated NO Production From
BAECs
The release of NO from control cells was 1.48±
0.28x10-4 and 6.09±0.42x10-4 pmol/cell
under basal conditions and in response to 1 µmol/L calcium ionophore
A23187, respectively. After incubation with 1 ng/mL TGFß1
for 24 hours, the release of NO under basal conditions and in the
presence of calcium ionophore A23187 was increased to
3.11±0.27x10-4 and 9.22±0.36x10-4
pmol/cell, respectively (Fig 2B).
TGFß1 Potentiated NOS Activity in BAECs
As assessed by arginine/citrulline conversion, NOS activity in
homogenates of TGFß1-treated cells (1 ng/mL
for 24 hours) was increased by 1.6 times that in control cells.
Conversion of L-[3H]arginine to
L-[3H]citrulline was inhibited by addition
of 10 µmol/L
NG-monomethyl-L-arginine,
an inhibitor of NOS, in both control and
TGFß1-treated cells (Fig 2C).
TGFß1 Increased the Transcriptional Activity of the
ecNOS Gene Promoter
Basal and TGFß1-stimulated (1 ng/mL for 16 hours)
promoter activity was assessed by luciferase assay. As shown in Fig 3, ecNOS -2835/+240, ecNOS -1548/+240, and ecNOS
-1269/+240 promoter activities were increased more than fourfold in
response to TGFß1 treatment. In contrast,
TGFß1 treatment did not alter transcriptional activities
of poLuc, pSV2Luc, ecNOS -935/+240, ecNOS -614/+240, and ecNOS
-416/+240 constructs. These results indicate that the
TGFß1-response element was likely present between
nucleotides -935 and -1269 of the ecNOS promoter, in
which a putative NF-1 binding site exists (nucleotide
-1014).
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Nuclear Extracts From BAECs Bound to ecNOS Promoter Fragment
Containing the Putative NF-1 Binding Site in a Sequence-Specific
Manner
The deletional analysis described above and the previous
reports indicating a role of NF-1 in transcriptional regulation by
TGFß116 17 prompted us to further examine
possible interactions of the putative NF-1 binding site of the ecNOS
promoter with endothelial cell nuclear proteins. Gel
mobility shift assays were performed with a 29-bp
oligonucleotide corresponding to the sequence from
nucleotide -1006 to nucleotide -1034 of the
bovine ecNOS promoter (NOS promoter fragment) and a consensus NF-1
oligonucleotide. When nuclear extracts prepared from
BAECs were incubated with radiolabeled NOS promoter fragments, several
retarded bands were observed (lanes 1 and 9 in Fig 4).
Addition of excess unlabeled NOS promoter fragments competed formation
of a major band, indicated by the arrow, but random
oligonucleotides had no such effect (lanes 2, 4, and 10
in Fig 4). This competition was dependent on the concentration of
unlabeled NOS promoter fragments (lanes 1 to 5 in Fig 5). These findings indicated that this band was a
sequence-specific complex of the NOS promoter fragment with nuclear
proteins. When the consensus NF-1 oligonucleotide was
used as a radiolabeled probe, a major retarded band was observed (lanes
5 and 14 in Fig 4). This major band was a sequence-specific complex of
the consensus NF-1 oligonucleotide with nuclear
proteins, because this complex was competitively inhibited by excess
amounts of unlabeled NF-1 consensus oligonucleotides
but not by random nonsense oligonucleotides (lanes 7,
8, and 15 in Fig 4 and lanes 14 to 16 in Fig 5). To further
characterize the nuclear protein(s) involved in these interactions, a
rabbit polyclonal antibody against CTF/NF-1 was used in a gel mobility
shift assay. This antibody (designated 8199), which was raised against
the N-terminal half of CTF/NF-1 that contains the DNA-binding domain,
crossreacts with other proteins in the CTF/NF-1 family and other
proteins related to CTF/NF-1 if they contain a similar DNA-binding
domain. Antibody 8199 strongly reduced the electrophoretic mobility of
the specific complexes of nuclear extracts with not only the consensus
NF-1 oligonucleotide but also the NOS promoter
fragment, whereas preimmune rabbit serum did not (lanes 11, 12, 16, and
17 in Fig 4). These supershifted bands (indicated on the figure by an
arrow) were competed by excess amount of unlabeled
oligonucleotides (lanes 13 and 18 in Fig 4). These
results indicate that protein(s) bound to the NOS promoter fragment
have a structure closely related to proteins of the CTF/NF-1 family.
Interestingly, competition experiments indicated that protein(s) bound
to the NOS promoter fragment had low affinity for the consensus NF-1
oligonucleotide. Excess amounts of unlabeled consensus
NF-1 oligonucleotides only minimally competed the
binding of nuclear protein(s) with the NOS promoter fragment (lane 3 in
Fig 4 and lanes 10 to 13 in Fig 5). Conversely, unlabeled NOS promoter
fragments had also little effect on the binding of nuclear extracts
with the consensus NF-1 oligonucleotide (lane 6 in Fig 4 and lanes 6 to 9 in Fig 5).
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The binding activity of nuclear extracts prepared from TGFß1-treated cells (1 ng/mL for 16 hours) for the NOS promoter fragment was inconsistently increased compared with that of control cells (1.3-fold increase in three different experiments, as assessed by densitometry). TGFß1 treatment had no effect on the binding activity to radiolabeled consensus NF-1 oligonucleotides.
Site-Directed Mutagenesis of the NF-1 Binding Site
To further explore the significance of the putative NF-1 binding
site in transcriptional activation by TGFß1, we
introduced a 6-bp substitution into the core (nucleotides
-1026 to -1014) of the putative NF-1 binding site of the ecNOS
-1548/+240/luciferase construct (Fig 6A). Gel mobility shift assay
demonstrated that this substitution caused the loss of the specific
binding affinity. As shown in Fig 6B, excess amounts of unlabeled
6-bp–substituted NOS promoter fragments (NF-1 mutant NOS promoter
fragment) did not compete the specific binding of the wild-type NOS
promoter fragment with nuclear protein. In a transfection study with
the NF-1 mutant ecNOS promoter/luciferase construct, mutation of the
putative NF-1 binding site in the promoter/luciferase construct
markedly decreased the responsiveness to TGFß1,
although even in the mutant ecNOS promoter/luciferase construct
TGFß1 increased promoter activity 1.9-fold (Fig 6C).
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TGFß1 Inhibited DNA Synthesis and Cell
Growth
Because ecNOS expression of proliferating cells is increased
compared with quiescent cells,11 we sought to determine
whether the effect of TGFß1 on ecNOS was simply due to a
stimulation of cell growth. TGFß1 (0.1 to 10 ng/mL)
inhibited both cell growth assessed by cell counting and DNA synthesis
assessed by [methyl-3H]thymidine incorporation in a
dose-dependent manner. TGFß1 (1.0 ng/mL for 48 hours)
reduced thymidine incorporation and cell number by 68.7% and 80.8%,
respectively. The viability of TGFß1-treated cells was
greater than 97.3%, as assessed by trypan blue exclusion.
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Discussion |
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The present study demonstrates that TGFß1 increases expression of ecNOS associated with an increased enzyme activity of NOS and production of NO in endothelial cells. Transcriptional activity of the 5'-flanking promoter of the ecNOS gene was increased by TGFß1. Deletion analysis suggests that the TGFß1 response element is present between nucleotides -1269 and -935 from the first transcription start site, in which a putative NF-1 binding site exists. Mutation of the putative NF-1 binding site significantly decreased the responsiveness to TGFß1. Gel shift assays show that nuclear protein(s), immunologically similar to CTF/NF-1, bind to the putative NF-1 binding site in a sequence-specific manner.
The increase in ecNOS mRNA induced by TGFß1 could be caused either by an increased transcriptional rate or by decreased degradation of mRNA. The results of the present study demonstrate that this effect is probably due at least in part to increased ecNOS transcription. First, inhibition of mRNA transcription with actinomycin D completely prevented the increase in ecNOS mRNA by TGFß1. Second, promoter/luciferase constructs demonstrated a fourfold increase in ecNOS promoter activity in the presence of TGFß1. Taken together, these data strongly suggest that the increase in ecNOS mRNA by TGFß1 is mediated by increased mRNA transcription. The present study does not address the possibility that TGFß1 might increase ecNOS stability, but this possibility seems unlikely given the reported stability of ecNOS mRNA10 and the relatively short period of time necessary for TGFß1 to induce this effect.
Several mechanisms for gene transcription regulation by
TGFß1 have been proposed. TGFß1
activates transcription of the mouse 2(I) collagen
gene16 and the PAI-1 gene through NF-1.17
TGFß1 is also reported to increase transcription of its
own gene promoter by means of the AP-1 binding region.18
In studies of promoter/luciferase constructs we found that promoter
activity was increased by TGFß1 in constructs containing
a putative NF-1 binding site (-1269/+240), whereas the -935/+240
luciferase construct (which lacked a putative NF-1 site but contained a
putative AP-1 site) was not responsive to TGFß1. Mutation
of the putative NF-1 binding site markedly decreased
TGFß1 responsiveness. These results indicate that the
NF-1 binding site in the promoter region plays an important role in
transcriptional activation of the ecNOS induced by TGFß1
treatment.
NF-1 interacts with the consensus sequence
TGG(N)6-GCCAA, which is found in viral and
cellular genomes.24 A similar sequence (TGGTGTGACCCCA) is
found at nucleotide -1014 upstream from the first
transcription start site of the ecNOS.8 Because the second
half of the recognition sequence for NF-1 resembles the CCAAT box and
NF-1 has been proposed to be identical to CTF,25 NF-1 is
often referred to as CTF/NF-1. Recent data suggest that CTF/NF-1 is a
heterogeneous family of nuclear proteins. The diversity of
these proteins is in part due to alternative splicing of NF-1 gene
transcripts.26 In gel mobility shift assays, a polyclonal
antibody against CTF/NF-1 crossreacted with the protein(s) bound to the
putative NF-1 binding site of the ecNOS promoter, whereas they had low
affinity for consensus NF-1 oligonucleotides. These
results suggest that nuclear protein(s) involved in the interaction of
the putative NF-1 binding site of the ecNOS promoter have a structure
closely related to the DNA-binding domain of proteins in the CTF/NF-1
family, but they are probably not identical to NF-1. These findings are
similar to recent data indicating that the human 1(I) collagen gene
promoter contains a TGFß-activating element similar to the NF-1
binding region, which does not interact with NF-1.27
Whereas transcriptional activity of the ecNOS promoter by
TGFß1 treatment was increased fourfold as assessed by
transfection of promoter/luciferase constructs, the binding activity of
nuclear extracts in TGFß1-treated cells was only modestly
and inconsistently increased. From these findings, we speculate
that a TGFß1-responsive transcriptional factor(s) similar
to CTF/NF-1 probably participates in activating the ecNOS gene
transcription without enhancing the binding affinity. The present
findings are entirely analogous to those of the PAI-1 promoter in which
transcription is activated by TGFß1 by means of a
similar NF-1 site.17 In the cases of both PAI-1 and ecNOS,
gel mobility shift assay failed to demonstrate enhancement of the
binding affinity by TGFß1 treatment. It is possible that
TGFß1 may activate gene transcription by means of
NF-1–like factors already bound to the promoter. Alternatively, other
regions of the ecNOS promoter may also participate in transcriptional
activation by TGFß1. This possibility is strengthened by
the observation that even in the NF-1 mutant ecNOS/luciferase construct
some responsiveness to TGFß1 was preserved.
The present experiments also demonstrate an increase in ecNOS protein, NO production, and enzyme activity in endothelial cells treated with TGFß1. The increases in protein and NO production were in general less than the increase in mRNA. This is not surprising because levels of mRNA and protein are not always directly correlated and factors other than the level of ecNOS protein may modulate cell NO production (eg, ecNOS cofactors, intracellular Ca2+). Although the increase in cellular NO release may seem modest, it is important to note that even small increases in NO production may markedly decrease the tone of intact vascular segments.2 28 Thus, small increases in NOS activity and NO production caused by TGFß1 may importantly influence vascular function. Another consideration related to this issue is that TGFß1 dramatically increases the production of extracellular matrix proteins, which were measured in our protein determinations and used to normalize enzyme activity and to calculate the amount of protein loaded onto gels for Western blot analysis. Although this was an unavoidable experimental condition, it is conceivable that if these data could have been normalized for intracellular proteins only, the amount of increases in ecNOS would have been greater.
Recently our laboratory demonstrated that expression of ecNOS of preconfluent growing cells is increased compared with that of postconfluent quiescent cells.11 Thus, one explanation for the present findings is that TGFß1 might have a growth-stimulating effect. As previously observed,29 30 however, TGFß1 decreased both endothelial cell number and [3H]thymidine incorporation. This finding excludes the possibility that the TGFß1 effect was simply due to stimulation of cell growth. Furthermore, in preliminary experiments, TGFß1 increased both ecNOS mRNA and the enzyme activity in quiescent cells several days after confluence was reached.
In previous experiments, we observed that vessels from animals with diet-induced atherosclerosis produced larger quantities of NO than normal animals.31 Because TGFß1 is present in increased quantities in atherosclerotic lesions,13 our findings in the present study may in part explain the enhanced production of NO in atherosclerotic vessels.
It has previously been demonstrated that TGFß1 decreases
adhesiveness of neutrophils to endothelial cell
monolayers.32 More recent studies have shown that NO also
decreases neutrophil adhesion to endothelial cell
monolayers, probably by means of scavenging of superoxide
anions.33 34 It is interesting to speculate that an
increase in NO production by TGFß1 plays a role
in the antiadhesive effect of TGFß1. It has also been
demonstrated that pretreatment with TGFß1 preserves
endothelial function and has a cardioprotective effect
in rat hearts subjected to ischemia/reperfusion injury,
possibly by means of inhibition of superoxide
production.35 TGFß1 might have
beneficial effects in such pathophysiological
situations by increasing endothelial cell NO
production. A major source of TGFß1 in vivo is
-granules in platelets. It is interesting to speculate that
TGFß1 released from platelets might serve to increase
endothelial cell NO production through enhanced
ecNOS expression. This could serve as a mechanism whereby platelet
adhesion to endothelial cells could be modulated by
enhanced endothelial NO release that was mediated by
TGFß1 released from platelets.
In conclusion, TGFß1 upregulates the expression of ecNOS mRNA and protein as well as NO production and NOS activity in endothelial cells. This response is mediated by transcriptional activation of the ecNOS promoter. This study is the first report to demonstrate involvement of a growth factor in gene regulation of the "constitutively expressed" ecNOS.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL32717, HL39006, and HL48667 and a merit grant from the Veterans Administration. We are grateful to Dr Naoko Tanese (Department of Microbiology, New York University Medical Center) and Dr Robert Tjian (Howard Hughes Institute, Department of Biochemistry, University of California) for kindly providing us with antibody against CTF/NF-1. We appreciate the secretarial assistance of Cynthia M. Curry.
Received September 9, 1994; accepted May 8, 1995.
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References |
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