| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Pulmonary and Critical Care Division, Department of Medicine, New England Medical Center/Tupper Research Institute; and
* Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA
1Correspondence: Pulmonary and Critical Care Division, New England Medical Center, 750 Washington St., NEMC #257, Boston, MA 02111, USA. E-mail vthannickal{at}lifespan.org
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(an EGF receptor ligand) are
able to rapidly (within 5 min) induce the generation of intracellular
O2.- without detectable NADH oxidase activity
or extracellular H2O2 release. In contrast,
TGF-ß1 does not stimulate intracellular O2.-
production and the delayed induction of extracellular
H2O2 release is not associated with
O2.- production. Expression of
dominant-negative Ras (N17Ras) protein by herpes simplex virus-mediated
gene transfer blocks mitogen-stimulated intracellular
O2.- generation but has no effect on
TGF-ß1-induced NADH oxidase activation/H2O2
production. These results demonstrate that there are at least two
distinctly different ROS-generating enzymatic systems in lung
fibroblasts regulated by mitogenic growth factors and TGF-ß1 via
Ras-dependent and -independent mechanisms, respectively. In addition,
these findings suggest that endogenous production of ROS by growth
factors/cytokines may have different biological effects depending on
the primary reactive species generated and site of
production.—Thannickal, V. J., Day, R. M., Klinz, S. G., Bastien, M. C., Larios, J. M., Fanburg, B. L.
Ras-dependent and -independent regulation of reactive oxygen species by
mitogenic growth factors and TGF-ß1.
Key Words: superoxide anion • hydrogen peroxide • cell growth • fibroblasts
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2
3
4
5)
. In contrast to the relatively
high levels of ROS produced by the phagocytic NADPH oxidase for
microbicidal activity, ‘low-level’ generation of these redox-active
biomolecules in nonphagocytic cells may have a role in the
regulation of normal physiological processes or in cell signaling
(6
, 7)
. Several studies have also demonstrated that
exogenous addition of ROS may have either positive or negative effects
on cell proliferation (reviewed in ref 8
). But the reasons
for these differences in cellular growth responses to various
‘pro-oxidant’ states induced either by the endogenous growth
factor/cytokine-stimulated ROS production or by exogenous addition of
ROS are poorly understood.
The enzymatic source(s) and mechanisms of action of growth
factor/cytokine-generated ROS are largely unknown. All known protein
subunits of the phagocytic
O2.--generating NADPH oxidase
are not expressed in some nonphagocytic cells (9
, 10)
.
However, Suh et al. recently demonstrated that a homologue of
gp91phox, the catalytic subunit of the phagocytic
oxidase, is expressed in vascular smooth muscle cells and may have a
role in cell growth regulation through the formation of
O2.- (11)
. In
addition, similar to the phagocytic oxidase, the Rac1 GTP binding
protein has been shown to regulate growth factor/cytokine-induced ROS
production in fibroblasts (12
, 13)
.
Most earlier studies of growth factor/cytokine-stimulated ROS focused
primarily on changes in intracellular redox state by monitoring changes
in H2O2-mediated oxidation
of an intracellular fluorescent compound (1
, 2
, 4
, 5)
.
Since O2.- rapidly dismutates to
H2O2 in most biological
systems, it is unclear whether the primary reactive oxygen species
formed in response to ligand stimulation is
O2.- or
H2O2. Moreover, since
H2O2 (unlike
O2.-) is able to diffuse across
the plasma membrane, it is not known whether ROS may be generated
extracellularly prior to intracellular localization. Mills et al.
recently demonstrated that chemical scavenging of
O2.- inhibits EGF-induced
intracellular H2O2
production in PC12 cells, suggesting that
O2.- (with subsequent
dismutation to H2O2) may be
the primary reactive species generated by EGF (5)
. Direct
formation of O2.- has been
demonstrated in NIH-3T3 fibroblasts stably transfected with a
constitutively active isoform of p21Ras
(14)
, but not in response to mitogenic stimulation with
specific growth factors.
The p21Ras family of proto-oncogenes is well
recognized for its ability to transduce mitogenic signals from receptor
tyrosine kinases (reviewed in ref 15
). However, the
primarily growth inhibitory cytokine, transforming growth factor ß1
(TGF-ß1), has also been shown to activate Ras proteins in intestinal
epithelial cells where it appears to have a role in TGF-ß’s
anti-proliferative effect (16
, 17)
.
p21Ras has also been shown to induce senescence
in primary human diploid fibroblasts by altering intracellular levels
of ROS (18)
.
TGF-ß1 induces the generation of extracellular
H2O2 in various cell types
(3
, 19
, 20)
. We have demonstrated that this activity is
associated with the activation of a novel cell surface-associated
NADH:flavin:O2 oxidoreductase (referred to as
NADH oxidase) in human lung fibroblasts (3)
. In contrast
to the early, relatively short-lived production of ROS in response to
mitogenic growth factors, TGF-ß1-induced
H2O2 generation is delayed
(>4 h, requiring transcription/new protein synthesis) and is sustained
for several hours after stimulation (3)
. It has not been
determined whether O2.- is a
precursor to TGF-ß1-induced
H2O2 production or if
intracellular ROS production precedes extracellular release. Moreover,
it is not known whether p21Ras regulates
TGF-ß1-induced NADH oxidase
activity/H2O2 production.
The purpose of this study was to compare mitogenic growth factor- and TGF-ß1-induced generation of ROS in normal human lung fibroblasts with respect to the primary reactive species produced (O2.- vs. H2O2), the site of generation (intracellular vs. extracellular) and regulation by p21Ras. Based on the hypothesis that differences in the primary reactive species produced and the site of production may alter cellular responses by activating different signaling pathways, the effect of endogenous ligand-induced ROS production on the regulation of cell growth was also examined.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, and recombinant human PDGF-BB were obtained from R&D Systems
(Minneapolis, Minn.). Human basic fibroblast growth factor (FGF-2) was
from Promega Corp. (Madison, Wis.). All other reagents and inhibitors
were from Sigma.
Measurement of intracellular O2.-
production
To measure intracellular
O2.- production, we used a
modification of a previously described method using lucigenin-enhanced
chemiluminescence (14
, 21
, 22)
. This is based on the
reaction between reduced lucigenin and
O2.- (but not
H2O2) resulting in the
emission of light. We have modified the method to allow measurement of
O2.- in adherent cells without
the need for assaying cells in suspension or using cell homogenates.
Cultured IMR-90 cells were grown on 35 mm petri dishes to 90–100%
confluency, serum-starved for 24 h, and stimulated with various
growth factors for defined periods of time. The medium was then
replaced with 1 ml of serum-free RPMI 1640 medium without phenol red
and containing 1 mM lucigenin. Chemiluminescence was recorded
immediately by placing the petri dish on a platform directly into the
sample chamber of a TD-20/20 luminometer (Turner Designs, Sunnyvale,
Calif.). After a 3 s dark adaptation, consecutive readings were
taken over 15–30 s intervals with each reading integrated over 15 s. With this technique, a relatively steady-state chemiluminescent
signal was observed over at least a 4 min period (as shown in
Fig. 1A
). Specificity of the reaction for
O2.- was demonstrated by the
almost complete inhibition (>90%) of the luminescent signal in both
control and mitogen-stimulated cells by preincubating cells for 30 min
with Tiron (1 mM), an intracellular
O2.- scavenger, and partial
inhibition (
60%) by extracellular SOD (100 U/ml), but not by
catalase (100 U/ml). For determination of a single value, the average
of 8 readings over a 2–4 min period was taken and the mean ±
SD of several experiments representing identically treated
dishes (minimum n=4) was used to plot data.
|
Measurement of H2O2 release
H2O2 release from
cultured fibroblasts into the overlying medium was assayed using a
modification of the method of Ruch and co-workers (23)
.
This fluorometric method is based on the conversion of homovanillic
acid, a substituted phenolic compound, to its fluorescent dimer in the
presence of H2O2 and
horseradish peroxidase. After exposure to mitogens or TGF-ß1, all
cells were washed with Hank’s balanced salt solution (HBSS), pH 7.4,
and then incubated with a reaction mixture containing 100 µM
homovanillic acid, 5 U/ml horseradish peroxidase, type IV, and 1 mM
HEPES in HBSS without phenol red, pH 7.4. This solution was then
collected after a 1 h incubation, the pH adjusted to 10.0 with 0.1
M glycine-NaOH buffer, and fluorescence measured at excitation and
emission wavelengths of 321 and 421, respectively. Linearity of the
rate of H2O2 release was
established by measuring the amount of
H2O2 released at regularly
timed intervals over a 2 h period. All incubations of experimental
samples were made with control samples containing the reaction mixture
alone (i.e., without cells) to correct for any spontaneous dimerization
of homovanillic acid. The exact concentrations of
H2O2 of solutions used to
plot standard curves were determined spectrophotometrically at 240 nm
using an extinction coefficient of 43.6 M-1 x
cm-1. Preincubating these samples with catalase
(100 U/ml), but not SOD (100 U/ml), completely inhibited the
fluorescence to background levels confirming the specificity of this
reaction for H2O2.
Measurement of NADH oxidase activity
Measurements of NADH oxidase activity were made as described
previously (3)
. Cells were washed with RPMI media without
phenol red (pH 7.4) and then incubated with NADH (250 µM) in the same
medium for varying time intervals. The rate of NADH consumption was
monitored by the decrease in absorbance at 
= 340 nm, using a
Hewlett-Packard 8452A diode array spectrophotometer. The absorption
extinction coefficient used to calculate the amount of NADH consumed
was 6.22 mM-1 x cm-1.
For measurements of specific NADH:flavin oxidoreductase activity, the
rate of NADH consumption inhibitable by diphenyliodonium (DPI), a
flavoprotein inhibitor (24)
, was used. This was done by
adding DPI (10 µM) 30 min prior to the assays for NADH consumption.
This ‘DPI-inhibitable’ NADH consumption was used as a measure of NADH
oxidase activity. All measurements were expressed in nanomoles of
NADH/min/106 cells.
Assessment of cell proliferation
Cell proliferation in response to mitogenic growth factors and
TGF-ß1 was assessed directly by cell counting in a Coulter counter.
Briefly, cells grown on 35 mm petri dishes as described above were
gently washed with phosphate-buffered saline, incubated with 1.0 ml of
trypsin-EDTA for 2–3 min, and rapidly suspended in solution by
pipetting. A 0.2 ml aliquot of cell suspension was then diluted in 20
ml of isotone solution prior to counting in a model ZM Coulter counter
(Coulter Electronics, Hialeah, Fla.). The cell number/dish was then
calculated based on a dilution factor that was identical for all
groups.
HSV-mediated transfer of dominant-negative Ras
A cDNA encoding a mutant Ras protein (N17Ras) (25)
was ligated and cloned into the herpes simplex virus (HSV) amplicon
HSVPrpUC to generate recombinant plasmids. A plasmid clone encoding
ß-galactosidase (ßGAL) in the same HSV amplicon was obtained from
Dr. Rachael Neve (Harvard Medical School, Boston, Mass.). Both plasmids
were packaged into virus particles in 2–2 cell line using the HSV
helper virus, 5dl1.2, according to published protocols
(26)
to yield the viral vectors HSV.N17Ras and HSV.ßGAL.
To optimize infection rates, IMR-90 cells were infected with various
dilutions of HSV.ßGAL (108 pfu/ml) and the
efficiency of protein expression was determined by staining for
ß-galactosidase activity. Almost 100% of cells showed some
expression of ß-galactosidase by this technique when cells were
infected with a mean of infection of 5–10 viruses/cell (Fig. 2C
).
|
For expression of dominant-negative Ras, IMR-90 cells deprived of serum
for 24 h were infected with HSV.N17Ras and HSV.ßGAL for a period
of 16 h prior to measurements for
O2.- and
H2O2 as described above. To
verify adequate expression of dominant-negative Ras protein, duplicate
lysates of infected cells were run on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and Western blotting was
performed as described previously (27)
. Immunoblotting for
Ras protein was with a polyclonal antibody raised against the entire 21
kDa human Ras (Ha-ras) protein corresponding to residues
1–190 (Transduction Laboratories, Lexington, Ky.)
Statistical analysis
Data from the various groups were expressed as means ±
SD. Statistical comparisons were made using the Student’s
t test for unpaired samples. For studies involving more than
two groups, two-way analysis of variance was determined using the
Scheffe’s test (GB-STAT: Dynamic Microsystems, Silver Spring, Md.).
Statistical significance in all cases was defined as P <
0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
shows that cells treated with PDGF-BB (10 ng/ml) for 5 min produced an
twofold increase in luminescent signal over control (solvent used
for PDGF-BB in the same final concentration, 1 mg/ml of BSA in 4 mM HCl
diluted 1:1000). Maximal levels of
O2.- generation in response to
PDGF-BB were observed 5–10 min after stimulation with a steady decline
to control levels by 45 min (results not shown). Other mitogenic growth
factors that bind RTKs, TGF- (20 ng/mlx5 min) and FGF-2 (10
ng/ml x5 min) also demonstrated significant increases in
O2.- production shortly after
ligand binding (Fig. 1B
).
To determine whether mitogenic growth factors stimulated extracellular
H2O2 release, cells treated
with PDGF-BB (10 ng/ml), FGF-2 (10 ng/ml), and TGF- (20 ng/ml) for
short (5 min) and longer durations (16 h) were assayed for NADH oxidase
activation/H2O2 release.
None of these growth factors stimulated any detectable
H2O2 release either at 5
min (Table 1
) or 16 h (results not shown). Concomitantly, there was no increase
in cell surface-associated NADH oxidase activity after stimulation with
these mitogens (Table 1)
.
|
TGF-ß1 induces delayed extracellular H2O2
release without O2.- production
We had previously demonstrated the ability of TGF-ß1 to induce
the generation of extracellular
H2O2 by a cell
surface-associated NADH oxidase (3)
. To determine whether
O2.- is formed as an
intermediate, with subsequent dismutation to
H2O2, we measured
O2.- production at the same time
point as the observed maximal generation of
H2O2. No elevation in
O2.- production was observed at
16 h after stimulation with TGF-ß1 (Fig. 1B
).
Moreover, addition of SOD (100 U/ml) did not alter the rate of
TGF-ß1-stimulated H2O2
release (results not shown). These results suggest that
O2.- is not the primary product
of the TGF-ß1-activated NADH oxidase and that
H2O2 may be formed directly
by 2-electron reduction of O2 to
H2O2.
We also examined the possibility that TGF-ß1 may activate an
O2.--generating oxidase similar
to that activated by mitogenic growth factors. When IMR-90 cells were
exposed to TGF-ß1 for 5 min, no increase in
O2.- production was observed
(Fig. 1B
), suggesting that immediate postreceptor signaling
by TGF-ß1 does not lead to intracellular ROS production similar to
the response induced by the RTK(s)-linked mitogenic growth factors.
TGF-ß1-induced extracellular H2O2 release
is not mediated by lysyl oxidase
Since the extracellular generation of
H2O2 was found to be
specific to TGF-ß1, we examined the possibility that this may be due
to a secondary effect of this cytokine related to its known effect on
the up-regulation of lysyl oxidase (28)
. Lysyl oxidase is
an extracellular, matrix-embedded, collagen cross-linking protein that
forms H2O2 as a by-product
of the oxidative deamination of amines to aldehydes (29)
.
ß-Aminopropionitrile and ethylenediamine are potent inhibitors of
lysyl oxidase (30)
. Neither ß-aminopropionitrile
(10-4 M) nor ethylenediamine
(10-4 M) had an effect on TGF-ß1-induced NADH
oxidase activation/H2O2
production (Table 1)
.
Ras is required for mitogen-stimulated O2.-
production but not for TGF-ß1-induced H2O2
production
To study the regulation of mitogen-stimulated
O2.- production and
TGF-ß1-induced H2O2
production by Ras, we infected IMR-90 cells with HSV vectors encoding a
dominant-negative isoform of p21Ras (HSV.N17Ras)
and ß-galactosidase (HSV.ßGAL, used as control). We were able to
achieve high efficiency of gene transfer and protein expression with
this technique. Figure 2C
shows almost 100% staining of
cells for ß-galactosidase activity after incubation with HSV.ßGAL
for 16 h. High-level expression of immunoreactive Ras protein by
Western blotting was observed when cells were infected for the same
duration with HSV.N17Ras (Fig. 2D
).
Cells overexpressing N17Ras protein failed to show an increase in
intracellular O2.- production in
response to PDGF (10 ng/mlx5 min) whereas control cells (HSV.ßGAL)
had a normal response (Fig. 2A
). This suggests that
functionally active Ras protein is required for
O2.- production in response to
mitogen stimulation. In contrast, TGF-ß1-induced extracellular
H2O2 production was not
inhibited by overexpression of dominant-negative Ras. The increase in
H2O2 production induced by
TGF-ß1 over unstimulated cells was similar in both the HSV.ßGAL and
HSV.N17 groups (Fig. 2B
). Of interest was the finding that
both HSV.ßGAL- and HSV.N17-infected cells had a higher baseline
release of extracellular
H2O2 than noninfected
cells, which may be related to a nonspecific effect of HSV infection.
Mitogen-stimulated ROS production is required for cell
proliferation but TGF-ß1-induced extracellular
H2O2 is not directly mitogenic
Growth-arrested fibroblasts stimulated with PDGF-BB (10 ng/ml),
FGF-2 (10 ng/ml), and TGF- (20 ng/ml) demonstrated significant
mitogenic capacity when cell numbers were assessed 48 h after
treatment by direct cell counting using a Coulter counter (results not
shown). PDGF-BB induced a 47% increase in cell growth, which was
significantly inhibited by pretreatment with SOD (100 U/ml added 30 min
prior to mitogen) whereas catalase (100 U/ml) had no significant effect
(Fig. 3
). TGF-ß1 (2 ng/ml) did not stimulate growth of these cells under the
same conditions and the anti-oxidant enzymes, SOD and catalase, had no
significant effects on cell growth (Fig. 3)
.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 7
). The
enzymatic source(s) and mechanisms of ROS generation in nonphagocytic
have not been clearly identified. In this study, we show that two
different ROS-generating enzymatic systems may be present in the same
cell and respond to mitogenic growth factors and TGF-ß1 by different
regulatory mechanisms (see schematic diagram, Fig. 4
).
|
The phagocytic NADPH oxidase is the best characterized of the plasma
membrane-associated oxidases and serves a specialized function in host
defense against invading microorganisms (reviewed in ref
31
). This multicomponent enzyme catalyzes the one-electron
reduction of O2 to
O2.- using NADPH as the electron
donor through the transmembrane protein subunit, cytochrome
b558. The transfer of electrons occurs
from NADPH on the inner aspect of the plasma membrane to
O2 on the outside. During phagocytosis, the
plasma membrane is internalized as the wall of the phagocytic vesicle
with what was once the outer membrane surface now facing the interior
of the vesicle. This targets the delivery of
O2.- and its reactive
metabolites internally for localized microbicidal activity
(31)
.
An enzyme similar to the NADPH oxidase of phagocytic cells has been
proposed as the source of growth factor-stimulated intracellular ROS
production in nonphagocytic cells (2
, 14)
. Due to the
ability of O2.- to rapidly
dismutate to H2O2, the
generation of O2.- in biological
systems is almost always accompanied by the formation of
H2O2 (32)
. In
fact, the ability of phagocytes to release extracellular
H2O2 from activation of the
O2.--generating NADPH oxidase
has been demonstrated (33)
. In this study, however,
extracellular H2O2
production was not detected in response to mitogens, suggesting that
mitogen-stimulated ROS is generated within an intracellular
compartment.
The peroxidase-based enzymatic assay for extracellular
H2O2 used in our studies
has been shown to be highly sensitive and specific for this reactive
species (34)
. We are able to detect
H2O2 concentrations in the
range of 10-8 M using this method that are at
least partly related to the high reactivity of
H2O2 with horseradish
peroxidase (104 times greater than with catalase)
(35)
. However, the measurement of intracellular ROS in
biological systems can be more problematic. Most methods are based on
the reaction of ROS with various ‘detector’ molecules that are
oxidatively modified to elicit luminescent or fluorescent signals. Two
of the more commonly used assays, the oxidation of
2',7'-dichlorofluorescin to fluorescent 2',7'-dichlorofluorescein and
lucigenin-enhanced chemiluminescence, have been reported to be
unreliable due to the potential for redox cycling of these compounds,
even in the absence of ROS (36
37
38)
. In our studies, assay
medium containing lucigenin alone (i.e., without cells) showed minimal
background luminescence suggesting that redox cycling of lucigenin in
solution in the absence of cells was insignificant. Unstimulated cells
demonstrated a significant level of luminescence consistent with a
baseline level of O2.-
production, which increased by two- to threefold in response to
mitogenic stimulation. In addition, there was a marked difference in
the chemiluminescent signal elicited from mitogen-stimulated cells vs.
TGF-ß1-treated cells, supporting a growth factor-specific effect and
making nonspecific reactions of lucigenin unlikely. Moreover, Irani et
al. reported that lucigenin-enhanced chemiluminescence in NIH 3T3
fibroblasts correlated well with a spin trap-dependent electron
paramagnetic resonance spectra that was strongly suggestive of the
generation of O2.- in response
to mitogenic stimulation (14)
.
In contrast to the shared mechanism of univalent reduction of
O2 to form
O2.- by the phagocytic NADPH
oxidase and the mitogen-activated ROS-generating enzymatic system,
TGF-ß1 generates extracellular
H2O2 without
O2.- formation, suggesting a
direct 2-electron transfer reaction. We have previously shown that
TGF-ß1-induced H2O2
production is associated with the activation of a cell
surface-associated flavoenzyme (NADH oxidase) (3)
. This
TGF-ß1-regulated oxidase appears to use a reaction mechanism common
to the class of flavoprotein oxidases that reduce
O2 directly to
H2O2, whereas electron
transferases generally mediate single electron transfers resulting in
the formation of O2.-
(39)
. We have eliminated the
H2O2-generating
extracellular matrix protein, lysyl oxidase, as the source of
TGF-ß1-induced extracellular
H2O2 production based on
the finding that two different inhibitors of lysyl oxidase had no
effect on H2O2 production.
Unlike NADH oxidase, lysyl oxidase does not require flavins as
cofactors for enzymatic activity (40)
and is not inhibited
by the flavoenzyme inhibitor, diphenyliodonium (H. M. Kagan,
personal communication).
The small GTP binding proteins Rac1 and p21Ras
have been shown to regulate the production of reactive oxygen species
in NIH 3T3 cells (12
, 14)
. Our results demonstrating that
several mitogenic growth factors (PDGF-BB, FGF-2, TGF-) stimulate
intracellular O2.- production in
lung fibroblasts via a Ras-dependent mechanism suggest that oncogenic
p21Ras may mediate a common signaling pathway
from a number of receptor tyrosine kinases (RTKs) leading to the
activation of an O2.--generating
oxidase/electron transferase (see schematic in Fig. 4
).
Unlike mitogenic growth factors that primarily signal via RTKs,
TGF-ß1 activates a heterotrimeric complex of type I/type II
transmembrane serine-threonine receptor kinases leading to the
phosphorylation/activation of the Smad proteins, which primarily
mediate growth-inhibitory signals and function as tumor-suppressor
genes (41)
. TGF-ß1 has been shown to activate Ras
proteins in intestinal epithelial cells where it appears to have a role
in mediating its anti-proliferative effect (16
, 17)
. Our
results show that, in contrast to mitogenic growth factors,
TGF-ß1-induced NADH oxidase
activation/H2O2 production
does not require p21Ras.
Based on the observed differences in ROS-generating enzymatic systems
(reaction mechanism and site of ROS production) and regulatory
mechanisms (requirement for p21Ras), it is likely
that these ligand-stimulated oxidase(s)/electron transferase(s) are
distinct. Recent work has demonstrated the presence of an
O2.--generating homologue of the
catalytic subunit of the phagocytic NADPH oxidase
gp91phox in vascular smooth muscle cells
(11)
. This supports the general concept that a family of
NAD(P)H-dependent oxidoreductases may be present in nonphagocytic cells
that function as generators of ‘redox signals’ in response to various
growth factors.
The finding that RTK(s)-linked growth factors require formation of ROS
to promote mitogenic signaling agrees with that of others (2
, 4)
. The observation that SOD is more effective than catalase in
inhibiting PDGF-stimulated cell growth is consistent with a reactive
oxygen species-specific effect. This also agrees with a recent study
that demonstrated that O2.- is a
kinetically more efficient and chemically more specific oxidant than
H2O2 for inactivating
protein-tyrosine phosphatase 1B (42)
. These enzymes
contain redox-sensitive cysteine groups in their active centers and may
function as a critical regulatory component in ROS-mediated mitogenic
signaling (43)
. It is difficult to draw conclusions
regarding the compartmentalization of mitogen-stimulated ROS based on
the inhibition of cell growth by antioxidant enzymes since their
ability to access the intracellular compartment is unclear. The
inhibition of mitogen-stimulated cell growth by SOD suggests that the
extracellularly added enzyme is at least partly able to access the site
of mitogen-stimulated O2.-. The
observation that fibroblast proliferation was not stimulated in
response to TGF-ß1-stimulated extracellular
H2O2 release suggests that
when generated at the cell surface,
H2O2 is either unable to
directly activate a mitogenic signaling pathway or that an independent
effect of TGF-ß1 is able to inhibit this pathway. The potential for
cell-derived H2O2 to
‘target’ neighboring cells in a paracrine manner in vivo
or to alter extracellular matrix proteins requires further study.
In summary, we have demonstrated that there are at least two distinctly different ROS-generating enzymatic systems present in human lung fibroblasts that respond to mitogenic growth factors and TGF-ß1 by Ras-dependent and -independent mechanisms, respectively. These findings also suggest that endogenous production of ROS by growth factors/cytokines may have different biological effects based on the primary species produced and the cellular microenvironment in which they are generated. Future studies in this emerging field of research will undoubtedly provide new insights on the roles of O2.-/H2O2 in mediating redox-regulated changes in protein structure/function essential for growth factor signaling and for the regulation of normal physiological processes.
|
ACKNOWLEDGMENTS |
|---|
Received for publication October 5, 1999.
Revision received January 28, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. R. Dobis, R. T. Sawyer, M. M. Gillespie, J. Huang, L. S. Newman, L. A. Maier, and B. J. Day Modulation of Lymphocyte Proliferation by Antioxidants in Chronic Beryllium Disease Am. J. Respir. Crit. Care Med., May 1, 2008; 177(9): 1002 - 1011. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. K. Vayalil, K. E. Iles, J. Choi, A.-K. Yi, E. M. Postlethwait, and R.-M. Liu Glutathione suppresses TGF-beta-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1281 - L1292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hatziapostolou, C. Polytarchou, P. Katsoris, J. Courty, and E. Papadimitriou Heparin Affin Regulatory Peptide/Pleiotrophin Mediates Fibroblast Growth Factor 2 Stimulatory Effects on Human Prostate Cancer Cells J. Biol. Chem., October 27, 2006; 281(43): 32217 - 32226. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sturrock, B. Cahill, K. Norman, T. P. Huecksteadt, K. Hill, K. Sanders, S. V. Karwande, J. C. Stringham, D. A. Bull, M. Gleich, et al. Transforming growth factor-beta1 induces Nox4 NAD(P)H oxidase and reactive oxygen species-dependent proliferation in human pulmonary artery smooth muscle cells Am J Physiol Lung Cell Mol Physiol, April 1, 2006; 290(4): L661 - L673. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hu, S. P. RamachandraRao, S. Siva, C. Valancius, Y. Zhu, K. Mahadev, I. Toh, B. J. Goldstein, M. Woolkalis, and K. Sharma Reactive oxygen species production via NADPH oxidase mediates TGF-{beta}-induced cytoskeletal alterations in endothelial cells Am J Physiol Renal Physiol, October 1, 2005; 289(4): F816 - F825. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Cheng, M. M. D. Encarnacion, G. M. Warner, C. E. Gray, K. A. Nath, and J. P. Grande TGF-{beta}1 stimulates monocyte chemoattractant protein-1 expression in mesangial cells through a phosphodiesterase isoenzyme 4-dependent process Am J Physiol Cell Physiol, October 1, 2005; 289(4): C959 - C970. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B. de Haan, N. Stefanovic, D. Nikolic-Paterson, L. L. Scurr, K. D. Croft, T. A. Mori, P. Hertzog, I. Kola, R. C. Atkins, and G. H. Tesch Kidney expression of glutathione peroxidase-1 is not protective against streptozotocin-induced diabetic nephropathy Am J Physiol Renal Physiol, September 1, 2005; 289(3): F544 - F551. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Mata-Greenwood, A. Grobe, S. Kumar, Y. Noskina, and S. M. Black Cyclic stretch increases VEGF expression in pulmonary arterial smooth muscle cells via TGF-{beta}1 and reactive oxygen species: a requirement for NAD(P)H oxidase Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L288 - L289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Ziel, V. Grishko, C. C. Campbell, J. F. Breit, G. L. Wilson, and M. N. Gillespie Oxidants in signal transduction: impact on DNA integrity and gene expression FASEB J, March 1, 2005; 19(3): 387 - 394. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. P. Brandes and J. Kreuzer Vascular NADPH oxidases: molecular mechanisms of activation Cardiovasc Res, January 1, 2005; 65(1): 16 - 27. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Paek, J. Park, and K.-J. Lee Multi-layered Representation for Cell Signaling Pathways Mol. Cell. Proteomics, October 1, 2004; 3(10): 1009 - 1022. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F.-S. Wang, C.-J. Wang, Y.-J. Chen, P.-R. Chang, Y.-T. Huang, Y.-C. Sun, H.-C. Huang, Y.-J. Yang, and K. D. Yang Ras Induction of Superoxide Activates ERK-dependent Angiogenic Transcription Factor HIF-1{alpha} and VEGF-A Expression in Shock Wave-stimulated Osteoblasts J. Biol. Chem., March 12, 2004; 279(11): 10331 - 10337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R.-M. Liu, Y. Liu, H. J. Forman, M. Olman, and M. M. Tarpey Glutathione regulates transforming growth factor-{beta}-stimulated collagen production in fibroblasts Am J Physiol Lung Cell Mol Physiol, January 1, 2004; 286(1): L121 - L128. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Gu, D. Weihrauch, K. Tanaka, J. P. Tessmer, P. S. Pagel, J. R. Kersten, W. M. Chilian, and D. C. Warltier Reactive oxygen species are critical mediators of coronary collateral development in a canine model Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1582 - H1589. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Esposito, G. Chirico, N. M. Gesualdi, I. Posadas, R. Ammendola, T. Russo, G. Cirino, and F. Cimino Protein Kinase B Activation by Reactive Oxygen Species Is Independent of Tyrosine Kinase Receptor Phosphorylation and Requires Src Activity J. Biol. Chem., May 30, 2003; 278(23): 20828 - 20834. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-P. Hammes, J. Lin, O. Renner, M. Shani, A. Lundqvist, C. Betsholtz, M. Brownlee, and U. Deutsch Pericytes and the Pathogenesis of Diabetic Retinopathy Diabetes, October 1, 2002; 51(10): 3107 - 3112. [Abstract] [Full Text] [PDF |
