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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 27, 2001 as doi:10.1096/fj.00-0572fje. |
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Department of Biochemistry, Osaka University Medical School, Osaka 565-0871, Japan; and
* Department of Biochemistry, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan
2Correspondence: Department of Biochemistry, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: proftani{at}biochem.med.osaka-u.ac.jp
SPECIFIC AIMS
This study addresses the hypothesis that nitric oxide (NO) up-regulates the heparin binding epidermal growth factor (EGF)-like growth factor (HB-EGF) through the accumulation of peroxide via inactivation of glutathione peroxidase (GPx) and the activation of JNK and that the induction of HB-EGF by NO serves as an autocrine protective factor against apoptosis by NO.
PRINCIPAL FINDINGS
1. Oxidative stress leads to the up-regulation of HB-EGF gene
expression
Heparin binding EGF-like growth factor (HB-EGF), a member of the
EGF family produced in smooth muscle cells (SMC) and the macrophages of
atherosclerotic plaques, plays an important role in atherogenesis.
Previous studies in our laboratory have shown that hydrogen peroxide
and reactive dicarbonyl metabolites, including methylglyoxal (MG) and
3-deoxyglucosone (3-DG), induce HB-EGF in rat aortic SMC (RASMC) via
the induction of oxidative stress. Vascular endothelial growth factor
(VEGF) induces HB-EGF in vascular endothelial cells and promotes a
50-fold increase in NO formation. On the basis of these
observations, we focused our attention on whether NO up-regulates
HB-EGF gene expression.
2. Induction of HB-EGF mRNA by nitric oxide in rat aortic smooth
muscle cells
The effect of SNAP on HB-EGF gene expression was examined by
Northern blot analyses. SNAP (100 µM) was found to induce HB-EGF mRNA
in a time-dependent manner (Fig. 1A
). When RASMC were incubated with various concentrations of
SNAP for 1 h or peroxynitrite for 2 h, the minimum
concentration required for HB-EGF mRNA up-regulation was observed to be
0.1 µM of SNAP and 100 µM of peroxynitrite, respectively (Fig. 1B
, C
). To better understand the mechanism by which NO
induces HB-EGF, the relationship between GPx and its inactivation by
NO, and HB-EGF induction were investigated. Since it is known that NO
readily reacts with thiol groups and a variety of molecules contain
free thiol groups that are capable of scavenging NO, thus abolishing
its effects in cells, we examined GPx activity in RASMC after treatment
with 100 µM of SNAP. After 1 h, GPx activity in SNAP-treated
cells was reduced by 71.2% compared with control cells. In addition,
the levels of intracellular peroxides in RASMC treated with SNAP
increased, as evidenced by flow cytometric analyses using a
peroxide-sensitive dye, H2DCF-DA. Because
peroxides (whose levels are increased as the result of the inactivation
of GPx) may act as a secondary signal molecule for NO, the effect of
catalase on SNAP-induced gene expression in RASMC was examined.
Catalase also blocked HB-EGF gene expression by 100 µM SNAP. To
further examine the issue of whether JNK activation modulates HB-EGF
gene expression by NO, plasmids containing the dominant-negative mutant
of JNK1 (T183A/Y185F) were transfected into the cells. When the
dominant-negative mutant of JNK1 was transfected into RASMC, the
expression of HB-EGF induced by 100 µM SNAP was decreased compared
with that of a mock transfectant or JNK1 wild-type. These results
strongly suggest that NO activates JNK through an increase in the
oxidation state of the cell, namely, as the result of increased
peroxide levels via the inactivation of GPx, thus regulating HB-EGF
gene expression.
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The effects of NO on the TNF-
-mediated apoptosis
NO is inherently cytotoxic, but peroxides, the levels of
which are increased as the result of inactivation of GPx, are also
contributors. Since TNF-+actinomycin (AcD) toxicity in cells is
associated with the induction of apoptosis, the effect of SNAP
pretreatment on TNF-+AcD-induced DNA fragmentation was examined.
Pretreatment with 0.1–10 µM SNAP 2 h before the addition of
TNF-+AcD led to a significant increase in the degree of DNA
fragmentation compared with that observed for TNF-+AcD alone. These
results suggest that although the concentration of NO is low, cellular
damage might well occur due to an increase in peroxide levels or the
inherent toxicity of NO.
An anti-apoptotic effect of HB-EGF
In the Western blot analyses, membrane-bound HB-EGF
protein levels were increased markedly by the presence of 100 µM SNAP
at 15 h. To determine whether the HB-EGF protein, which is induced
by SNAP, could result in adaptive increase in terms of resistance to
TNF-+AcD-induced DNA fragmentation, RASMC were pretreated with
concentrations of SNAP ranging from 0 to 100 µM for 5 or 15 h,
then treated with TNF-+AcD for 24 h. When TNF-+AcD were
added 15 h after 100 µM SNAP treatment, the degree of DNA
fragmentation was reduced to a level consistent with HB-EGF induction.
To confirm whether NO-induced HB-EGF is involved in the prevention of
apoptosis in RASMC, an HB-EGF antisense s-oligonucleotide was added to
the SNAP-treated cells in serum-free media because the antisense
oligonucleotides reduce the production level of membrane-bound HB-EGF.
The spontaneous loss of viability after 48 h was reversed in an
NO-dependent manner in serum-free media and led to a reduction in the
levels of apoptosis after treatment with antisense HB-EGF
s-oligonucleotides. When RASMC were treated 100 µM or 1 mM of SNAP
for 48 h after pretreatment of antisense HB-EGF
s-oligonucleotides, the antisense HB-EGF s-oligonucleotides resulted in
a significant induction of DNA cleavage. With antisense HB-EGF
s-oligonucleotides and SNAP treatment, some nuclei exhibited typical
apoptotic characteristics, such as nuclear condensation and
fragmentation. Corresponding to these changes, the nuclei appeared
typically condensed and fragmented by DAPI staining. Using propidium
iodide to stain the permeabilized cells, we detected an increase of
apoptotic cells, which have the subdiploid-staining profile, in RASMC
treated with 1 mM SNAP or SNAP+ antisense HB-EGF. These results suggest
that HB-EGF plays an important role in the anti-apoptotic process.
CONCLUSIONS
NO is an important signal transducer in many types of cells and tissues including blood vessels, neuronal, and hematopoietic cells. NO functions via a variety of mechanisms, including nitrosylation of (or redox reactions with) metal-and thiol-containing proteins. Our previous studies showed that GPx was inactivated by SNAP, an NO donor. These data suggest that NO directly inactivates GPx, resulting in an increase in intracellular peroxides, which in turn are responsible for cellular damage or gene expression. Even though SMC is a major target for NO, the effect of NO on the regulation of growth factors in SMC is not understood. In this study we demonstrate that NO up-regulates HB-EGF gene expression in SMC. This is the first report that demonstrates the stimulation of HB-EGF gene expression by NO in SMC. When SMC were treated with NO, levels of intracellular peroxides increased. An increase in peroxide levels by NO may play an important role in the regulation of apoptosis or gene expression. This raises the question of the nature of the mechanism by which intracellular peroxide levels are increased by NO. We focused on GPx inactivation by NO because GPx is an important antioxidant enzyme in the detoxification of H2O2. We demonstrate that NO-induced HB-EGF gene expression was blocked by NAC, a reducing agent known to alter the redox state of the cell. It is believed that the regulation of gene expression by NO is due to an increase in intracellular peroxides.
Recent studies showed that the rat HB-EGF gene promoter contains AP-1 sites. This implies that JNK activation by oxidative stress is important to HB-EGF gene expression. We have demonstrated here that NO induces JNK activation via an increase of intracellular peroxides. We also confirmed that JNK activation by NO is an important issue for HB-EGF gene expression using dominant-negative JNK mutants. These observations provide the first evidence for our understanding of the role of JNK in HB-EGF gene expression.
There is evidence that the membrane-bound form of HB-EGF promotes cell
survival. We recently showed that pro-HB-EGF-producing hepatoma cells
were resistant to apoptosis. NO regulates apoptosis and proliferation
in the vascular smooth muscle cells. Heat shock protein 70 expression,
which is induced by NO, inhibits TNF--induced apoptosis in rat
hepatocytes. It is therefore possible that HB-EGF expression by SNAP
may act as a survival factor in protecting against stress. When the
system was treated with antisense HB-EGF s-oligonucleotides, cell
viability decreased significantly as the result of the failure of
HB-EGF protective effects, which may otherwise cause the progress of
apoptosis by H2O2
due;F2> to NO-induced GPx inactivation (Fig. 2
).
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Endothelial cells appear to generate oxygen-derived free radicals under
pathological and physiological stimuli such as ischemia/reperfusion,
inflammation, and shear stress changes. ROS production by smooth muscle
cells is not as well characterized, although these compounds almost
exclusively enhance mitogenesis in these cells. The smooth muscle cell
functions as a final effector target for ROS, which is generated in a
paracrine manner by the endothelium or figurated blood elements. Even
though RASMC express superoxide dismutase (SOD) in order to provide
protection from NO toxicity, the inactivation of GPx may nevertheless
occur in certain cells that produce NO or in surrounding cells. This is
because other antioxidative enzymes, such as SOD or catalase, are not
inhibited by NO. The increased accumulation of peroxides within cells
after treatment with an NO donor is likely a consequence of the
inactivation of GPx by NO. An increase in peroxide levels may act as a
secondary signal molecule and a mediator of NO-mediated toxicity. In
fact, pretreatment with 0.1–10 µM SNAP for 2 h before the
addition of TNF-+AcD markedly increased the degree of DNA
fragmentation compared with TNF-+AcD alone. These results suggest
that an increase in peroxide levels through the inactivation of GPx by
NO affects various signaling cascades and that peroxide might be
involved in this toxic effect. Based on the results cited here, we
conclude that GPx inactivation by NO is one of the key mechanisms of
ROS production and its role in smooth muscle cells. In conclusion, our
data indicate that NO activates JNK via
H2O2 via the inactivation
of GPX by NO and induces HB-EGF, an autocrine protective factor in
RASMC.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0572fje ; to cite this
article, use FASEB J. (April 27, 2001) 10.1096/fj.00-0572fje 
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