Molecular Medicine |
From the Division of Cardiology, Emory University, Atlanta, Ga.
Correspondence to David G. Harrison, Division of Cardiology, Emory University, 1639 Pierce Dr, Atlanta, GA 30322. E-mail dharr02{at}emory.edu
| Abstract |
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-phenylnitrone, but it was inhibited by
the antioxidants N-acetylcysteine, ebselen, and
exogenously added catalase. Unlike H2O2, the
4.0-fold induction of eNOS by shear stress (15 dyne/cm2 for
6 hours) was not inhibited by N-acetylcysteine or
exogenous catalase. In conclusion, H2O2
increases eNOS expression through transcriptional and
post-transcriptional mechanisms. Although H2O2
does not mediate shear-dependent eNOS regulation, it is likely to be
involved in regulation of eNOS expression in response to other
physiological and/or pathophysiological
stimuli.
Key Words: paraquat superoxide dismutase eNOS mRNA stability cultured endothelial cells
| Introduction |
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A common property of many stimuli that increase eNOS expression is the ability to increase the production of reactive oxygen intermediates (ROIs) in endothelial cells. Thus, laminar shear stress,11 cyclic strain,12 oxLDL,13 high glucose,14 TGF-ß15 and proliferation16 have all been associated with an elevated production of ROIs in endothelial cells. Furthermore, for many of these stimuli, this increase in production of ROIs was shown to be important for regulating expression of other genes. For example, induction of heme-oxygenase-1,17 c-fos,18 monocyte chemotactic protein-1,19 and intercellular adhesion molecule-1 (ICAM-1)20 21 in human umbilical vein endothelial cells by shear stress and cyclic strain was inhibited by the antioxidants N-acetylcysteine (NAC) and catalase. Similarly, the phenolic antioxidants probucol and vitamin E have been shown to inhibit oxLDL-mediated induction of ICAM-1 and vascular cell adhesion molecule-1 expression.22 Finally, transcriptional induction of macrophage-colony stimulating factor by TGF-ß1 was inhibited by catalase but not by superoxide dismutase (SOD), suggesting that H2O2, rather than superoxide anions (O2-·), mediates this effect.23
These observations raise the possibility that ROIs represent common signaling molecules for modulating eNOS expression. Indeed, the human, bovine, and murine eNOS promoter regions contain putative binding sites for redox-sensitive transcription factors, including activator protein-1 (AP-1), Sp1, and antioxidant-responsive elements.24 25 26 Thus, the present study was performed to examine the effects of ROIs on eNOS expression in both BAECs and human aortic endothelial cells (HAECs). Our results indicate that H2O2, but not O2-· or hydroxyl radicals (OH·), increases eNOS expression by increasing both the rate of transcription of the eNOS gene and the stability of the eNOS message once it is formed. Finally, because shear stress has been shown to increase both eNOS expression and endothelial cell ROI production, additional studies were performed to determine whether ROIs might mediate increased eNOS expression in response to shear stress.
| Materials and Methods |
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Assessment of eNOS Expression
Northern analyses, Western blots, nuclear run-ons, and
eNOS enzyme activity assays were performed as previously
described.5
Drugs and Their Suppliers
Mn(III)tetrakis(4-benzoic acid)porphyrin chloride
(MnTBAP), N-tert-butyl-
-phenylnitrone (BPN),
5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole
(DRB), and ebselen were obtained from Calbiochem, and bovine liver
catalase was from Boehringer Mannheim. All other chemicals were
from Sigma. DMSO was used to dissolve BPN (1 mol/L) and DRB (25
mmol/L). MnTBAP (100 mmol/L) was dissolved in
NaHCO3 (1 mol/L). All other drugs were dissolved
in dH2O.
| Results |
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H2O2 Increases eNOS Expression and
Activity
The above results demonstrate that although eNOS expression is not
affected by O2-·, dismutation
of O2-· potently stimulates
eNOS expression. We therefore examined the effect of directly applied
H2O2. Treatment of BAECs
with 100 and 150 µmol/L
H2O2 for 24 hours resulted
in 3.1- and 5.2-fold increases in eNOS mRNA expression, respectively
(Figure 2
). The eNOS-inducing effect of
H2O2 (100 µmol/L)
was also time-dependent. After 6 hours of exposure to
H2O2, eNOS mRNA expression
was 1.6 times that in the time-matched controls. eNOS expression
continued to increase with time, reaching levels that were 3.1 and 3.6
times those in the time-matched 12- and 24-hour control groups,
respectively (Figure 2
).
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H2O2 also caused a
concentration-dependent increase in eNOS protein levels in BAECs as
measured by Western analyses. eNOS protein expression in BAECs
treated with 100 and 150 µmol/L
H2O2 was 2.1 and 2.8 times
that in control cells, respectively (Figure 3
). Likewise, eNOS enzyme activity, as
measured by conversion of
[14C]L-arginine to
[14C]L-citrulline, was increased by
H2O2 (Figure 3
).
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Similar to its effects on BAECs,
H2O2 also increased eNOS
protein expression in HAECs. In these cells, treatment with 100 and
200 µmol/L H2O2
caused 1.6- and 1.7-fold increases in eNOS protein expression,
respectively (Figure 4
).
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Effect of Short-Term Exposure to H2O2 on
eNOS Expression
Experiments were performed to determine the length of exposure
needed for H2O2 to
stimulate eNOS expression. BAECs were treated with
H2O2 (100 µmol/L)
for 0.25 to 12 hours. In all cases, cells were washed and exposed to
control medium and harvested at 12 hours after the beginning of the
H2O2 exposure.
H2O2 exposure as short as
0.25 hours caused a 4.4-fold increase in eNOS mRNA expression (Figure 5
). Maximal effects (ie, 8.3- and
7.8-fold increases compared with time-matched controls) occurred after
1- and 3-hour exposures (Figure 5
).
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Additional experiments were performed to determine how long eNOS
expression remained elevated after short-term
H2O2 exposure. Cells were
harvested 6, 12, 24, 48, and 72 hours after a 1-hour-pulse exposure to
H2O2 (100 µmol/L).
At all of the time points tested, eNOS mRNA expression was
significantly higher in
H2O2-treated cells than in
time-matched controls (Figure 5
). Maximum effects of
H2O2-pulse treatment
occurred after 12 and 24 hours (7.6 and 8.4 times control levels,
respectively; Figure 5
). However, even at 72 hours after the
1-hour H2O2 pulse, eNOS
mRNA expression remained elevated at 3.9 times that of control
levels (Figure 5
).
Effect of Scavengers on H2O2 Induction
of eNOS
Because the effects of
H2O2 could be mediated
either directly or indirectly by OH·, produced
from H2O2 via Fenton
chemistry, the effects of scavengers of
H2O2 and
OH· were examined. Induction of eNOS mRNA
expression by H2O2 was
abolished by catalase (1000 U/mL) and was significantly inhibited by
50% by the glutathione peroxidase mimetic ebselen (40
µmol/L) (Figure 6
). NAC (5
mmol/L), whether retained in the medium throughout the
H2O2 incubation or washed
out just before the addition of
H2O2, also abolished the
increase in eNOS mRNA expression caused by
H2O2 (Figure 6
).
None of these interventions affected basal eNOS mRNA expression (data
not shown). In contrast, the OH· scavengers
DMSO (0.3%), mannitol (20 mmol/L), and BPN (1 mmol/L) had no
effect on H2O2-induced eNOS
mRNA expression (Figure 6
). Moreover, cotreatment with
H2O2 (100 µmol/L)
and either of the Fenton chemistryenhancing agents, EDTA (100
µmol/L) or FeSO4 (100 µmol/L), was not
more effective at inducing eNOS mRNA expression than was
H2O2 alone (Figure 6
). Thus, these data indicate that the effect of
H2O2 on eNOS expression is
unlikely to be mediated by
OH·.
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Effect of H2O2 on eNOS Gene Transcription
and eNOS mRNA Stability
To examine the effect of
H2O2 on eNOS mRNA
transcription rate, nuclear run-on analyses were performed.
BAECs were treated with 100 µmol/L
H2O2 for 6 hours and nuclei
harvested. H2O2-treated
BAECs produced 3.0 times the amount of radiolabeled eNOS mRNA
transcripts of those made by control cells (Figure 7
). In contrast,
H2O2 had no effect on
transcription rate of the housekeeping gene, ß-actin (Figure 7
).
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To determine the effects of
H2O2 on eNOS mRNA
stability, BAECs were incubated with control or
H2O2 (100
µmol/L)containing medium for 6 hours. Next, cells from each group
were treated with the RNA polymerase II inhibitor DRB
(60 µmol/L). The rate of eNOS mRNA decay was significantly
slower in H2O2-treated
cells than it was in control cells (Figure 7
). In
H2O2-treated cells, the
half-life of the eNOS message was
20 hours compared with
7 hours
in control cells. Thus,
H2O2 increases eNOS
expression both by increasing the rate of transcription and by
elevating the half-life of eNOS mRNA.
Effect of H2O2 Scavengers on Shear-Induced
eNOS Expression
Shear stress has been shown to increase both eNOS expression and
production of ROIs by the endothelium. To
determine whether H2O2
produced in response to shear stress might mediate the increase in eNOS
expression, cells were exposed to a laminar shear of 15
dyne/cm2 for 6 hours. In the absence
of other interventions, shear caused a 4.0-fold increase in eNOS mRNA
expression. Addition of exogenous catalase or NAC had no effect on
induction of eNOS by shear (Figure 8
).
Thus, the increase in eNOS expression produced by shear stress is
unlikely to be caused by endogenously produced
H2O2.
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| Discussion |
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In recent years, it has become apparent that ROIs play an important role in gene regulation in response to several stimuli.28 These effects likely result from stimulation of intracellular phosphorylation cascades, leading to activation of transcription factors. In the present study, we demonstrated a 3-fold increase in transcription rate of the eNOS gene in response to H2O2. The 5'-promoters of the human, bovine, and murine eNOS genes contain putative binding sites for a number of redox-regulated transcription factors, including AP-1, Sp1, and antioxidant-responsive elements.24 25 26 In the present study, we did not examine the role of the 5'-promoter in the transcriptional induction of eNOS by H2O2. However, a previous study showed through gel-shift assays that the H2O2 generating system, glucose oxidase, increased binding of BAEC nuclear proteins to a radiolabeled probe containing an eNOS promoter AP-1 binding site.29 It is thus tempting to speculate that AP-1 and perhaps other redox-sensitive cis-acting elements are involved in the response to H2O2.
In addition to increasing eNOS gene transcription,
H2O2 also enhanced the
stability of the eNOS message. Modulation of eNOS mRNA stability is now
known to represent an important mechanism for regulation of
expression of the enzyme by a number of stimuli. For example, tumor
necrosis factor-
decreases eNOS expression by reducing the stability
of the eNOS message.30 In addition, stabilization of the
eNOS message augments eNOS expression in response to cell
growth9 and 3-hydroxy-3-methylglutarylCoA reductase
inhibitors.10 This effect of cell growth on
eNOS mRNA stability has been found to be due to decreased binding of a
51-kDa mRNA-destabilizing protein to a G+C-rich region near the start
of the 3'-untranslated region.9 Interestingly, preliminary
UV cross-linking studies in our laboratory showed that
H2O2 similarly decreased
binding of a 51-kDa protein to the eNOS mRNA 3'-untranslated
region.
Of note, in preliminary studies, we found that the duration of exposure to H2O2 required to elicit an increase in eNOS expression was substantially longer in HAECs than in BAECs. Although the explanation for this remains unclear, it may relate to the longer eNOS mRNA half-life in human30 as compared with bovine endothelial cells (ie, >24 hours versus 7 to 8 hours). Therefore, although a 24-hour exposure to H2O2 in BAECs is equivalent to 3 eNOS mRNA half-lives, it is only equal to 1 half-life in HAECs. Because at least half of the effects of H2O2 on eNOS are mediated by enhancing mRNA stability, longer exposures to H2O2 may be necessary to impact on steady-state eNOS mRNA levels in human cells.
Recently, we demonstrated that phenolic antioxidants, including NDGA and probucol, increased eNOS mRNA and protein expression.3 Although these findings may seem to be at odds with those of the present study, it is known that phenolic antioxidants can exert both antioxidant and pro-oxidant effects, depending on the prevailing redox environment. Abstraction of the hydrogen atom from the phenolic group of such antioxidants by O2-· results in formation of a phenoxyl radical (itself a pro-oxidant species) along with H2O2, which could in turn induce eNOS expression. Augmentation of the effects of NDGA by the reducing compounds NAC and ascorbic acid, observed in our previous study,3 could be explained if these compounds were acting to promote reduction of the phenoxyl radical back to the parent compound,31 thus allowing further participation in redox cycling.
Diverse stimuli, including cyclic strain, shear stress, and TGF-ß1, have been shown to induce endothelial cell expression of different genes in a manner that is abolished by NAC and catalase,17 18 19 20 21 23 implying that the upregulation is dependent on generation of endogenous H2O2. In these same studies, exogenous H2O2 in concentrations of 100 to 150 µmol/L induced a similar magnitude of expression of these genes. Thus, although the concentration of H2O2 used in this study and previous studies may seem high, these concentrations likely have physiological and pathophysiological significance. Because of intracellular catalase, glutathione peroxidase, and other antioxidants, the ultimate intracellular concentration of H2O2 is likely to be substantially lower than that added exogenously. Importantly, these concentrations of H2O2 exerted little or no microscopically visible toxic effects on endothelial cells.
To examine the physiological role of H2O2 in regulation of eNOS expression, we studied the effects of antioxidants on eNOS induction by shear stress. Several studies have shown that shear stress increases ROIs within the endothelium,8 11 18 20 probably via activation of an NADH-dependent oxidase.17 Furthermore, activation of heme-oxygenase-1,17 c-fos,18 and ICAM-120 by shear stress was inhibited by catalase and NAC, indicating that ROIs are likely to be important in induction of these genes. Additionally, we have shown that laminar shear stress induces Cu2+/Zn2+ SOD expression in BAECs,32 which should shift the balance of ROIs from O2-· to H2O2. We therefore hypothesized that eNOS induction by shear stress may be due to H2O2 production. This hypothesis was proven incorrect, however, as shear stress induction of eNOS was not inhibited by NAC or catalase, both of which abolished eNOS upregulation by H2O2. Moreover, preliminary studies in our laboratory demonstrate that the intracellular signaling pathways mediating the effects of H2O2 and shear stress on eNOS expression are different. For example, eNOS induction by shear stress seems to be inhibited by the c-Src inhibitor PP1. In contrast, H2O2-dependent eNOS induction is not blocked by this compound but can be inhibited by tyrphostin A25 and herbimycin A, implicating a role for a nonSrc-related tyrosine kinase in the transduction pathway.
Although H2O2 does not mediate the effect of shear stress on eNOS expression, it may be important in modulating eNOS expression in other settings. The production of O2-· by vascular cells is increased in several pathophysiological conditions, including hypertension, hypercholesterolemia, and diabetes. This increase in O2-· production is responsible for decreasing the bioactivity and thus beneficial actions of NO· in these disease states. Because O2-· is a precursor to other ROIs, including H2O2, it is likely that this response of increased eNOS expression represents an important compensatory mechanism. Indeed, in several pathological conditions, including early hypercholesterolemia,33 hypertension,34 and diabetes (T. Münzel, personal communication), eNOS expression is increased. It is therefore interesting to speculate that the increase in ROIs (especially H2O2) in these diseases underlies the increase in eNOS expression.
| Acknowledgments |
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Received July 8, 1999; accepted November 22, 1999.
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