(Received for publication, September 1, 1995)
From the
The cellular source(s) and mechanisms of generation of reactive
oxygen species (ROS) in nonphagocytic cells stimulated by cytokines are
unclear. In this study, we demonstrate that transforming growth factor
1 (TGF-
1, 1 ng/ml) induces the release of
H
O
from human lung fibroblasts within 8 h
following exposure to this cytokine. Elevation in H
O
release peaked at 16 h (
22 pmol/min/10
cells)
and gradually declined to undetectable levels at 48 h after TGF-
1
treatment. NADH consumption by these cells was stimulated by TGF-
1
while that of NADPH remained unchanged. NADH oxidase activity as
measured by diphenyliodonium (DPI)-inhibitable NADH consumption in
TGF-
1-treated cells followed a time course similar to that of
H
O
release. DPI, an inhibitor of the NADPH
oxidase complex of neutrophils and other flavoproteins, also inhibited
the TGF-
1-induced H
O
production.
Inhibitors of other enzymatic systems involving flavoproteins that may
be responsible for the production of H
O
in
these cells, including xanthine oxidase, nitric oxide synthase, and
both mitochondrial and microsomal electron transport systems, failed to
inhibit TGF-
1-induced NADH oxidation and H
O
production. The delay (>4 h) following TGF-
1 exposure
along with the inhibition of this process by cycloheximide and
actinomycin D suggest the requirement of new protein synthesis for
induction of NADH oxidase activity in TGF-
1-stimulated
fibroblasts.
Transforming growth factor (TGF-
), (
)a
potent fibrogenic cytokine, is overexpressed at sites of active tissue
repair and fibrosis such as in idiopathic pulmonary
fibrosis(1) , glomerulonephritis(2) , and vascular
restenosis(3) . Administration of anti-TGF-
antibodies has
been shown to suppress the in vivo activity of TGF-
1 and
protect against tissue fibrosis in kidney(4) ,
skin(5) , and lung(6) . Multiple actions of TGF-
on target cells appear to be important in tissue repair and remodeling
including its ability to regulate its own production as well as that of
other cytokines(7) . However, signaling mechanism(s) of this
and many other of its actions on target cells are poorly understood.
There is growing recognition that redox-active biomolecules play
important roles in cellular signaling, including activation of
transcription factors(8) . HO
, in
particular, is a powerful activator of NF-
B, a transcription
factor that is associated with gene activation of a number of cytokines
involved in tissue injury and repair processes(9) . Other
cytokines involved in these processes, namely TNF-
and IL-1, have
also been reported to stimulate production of ROS in nonphagocytic
cells(10) . Although the source of ROS generation in these
cells was initially thought to represent a plasma membrane-bound NADPH
oxidase similar to that of neutrophils, subsequent studies have
demonstrated this enzymatic complex to be structurally and genetically
unrelated(11) .
The ability of TGF-1 to stimulate
cellular production of H
O
, at least in some
cell lines, has been well established(12, 13) .
However, the source of this TGF-
1-stimulated ROS generation has
not been determined. We have previously reported that TGF-
1
stimulates the production of H
O
in endothelial
cells (12) . In the current study, we extend this observation
of a pro-oxidant effect of TGF-
1 to human lung fibroblasts. The
main purpose of this study was to identify the source(s) of the
TGF-
1-induced H
O
production in lung
fibroblasts and to specifically determine if NAD(P)H oxidoreductase
activities might be involved.
Figure 3:
Effect of
DPI on the rates of NADH and NADPH consumption by control and
TGF-1-treated cells. Measurements were made 16 h following
exposure to TGF-
1 (1 ng/ml). DPI (10 µM) was added 30
min prior to the assay, and rates were measured over a 2-h period.
Values are mean ± S.D., n =
4.
Figure 1:
Time course of the
rate of HO
release from lung fibroblasts
following TGF-
1 (1 ng/ml) treatment. There was no detectable
release of H
O
from control cells. Values are
mean ± S.D., n = 4.
Figure 2:
Rates of NADH and NADPH consumption by
control cells and cells treated with TGF-1 (1 ng/ml) for 16 h.
Values are mean ± S.D., n =
4.
We also examined the possibility
that NADH oxidation in the extracellular medium was due to
NADH-utilizing enzymes released into the medium of TGF-1-treated
cells. Conditioned media from control and TGF-
1-treated cells were
assayed for NADH oxidase activity (by measuring DPI-inhibitable NADH
consumption) and H
O
. Both NADH oxidase activity
and H
O
were undetectable in the conditioned
media of control and TGF-
1-treated cells. The rates of
DPI-noninhibitable NADH consumption were similar in both groups. The
requirement for the fibroblast monolayer to be in direct contact with
the extracellular medium for detection of NADH oxidase activity and
H
O
release suggests that these reactions are
taking place in or around the surface of fibroblast plasma membranes.
Measurement of NADH oxidase activity cannot, therefore, be accounted
for by release of enzymes into the extracellular medium.
Cycloheximide (1 µg/ml, an
inhibitor of protein synthesis) and actinomycin D (0.05 µg/ml, an
inhibitor of gene transcription) also produced significant reductions
in NADH consumption and HO
release, suggesting
that new protein synthesis is required for the NADH oxidase activity
induced by TGF-
1 in fibroblasts. Of uncertain significance is the
finding of a small amount of H
O
release by
TGF-
1-treated cells in the presence of cycloheximide and
actinomycin D despite complete inhibition of the TGF-
1-induced
increase in NADH consumption by these inhibitors.
We examined the
possibility that TGF-1-induced NADH oxidation may occur as a
result of a peroxidase-catalyzed free radical chain reaction in the
presence of H
O
and a transitional metal ion
(the ``peroxidase-oxidase'' reaction)(18) . Addition
of superoxide dismutase (Cu,Zn-superoxide dismutase, 100 units/ml) to
the reaction mixture did not inhibit TGF-
1-induced NADH oxidation
or H
O
release (Table 1), making this
reaction unlikely. Nonenzymatic oxidation of NADH by
H
O
was also ruled out by adding
H
O
(1-10 µM, concentrations
that exceeded experimentally measured levels) directly to the NADH
reaction mixture and finding no significant change in absorption at
= 340 nm.
Figure 4:
Time
course of NADH oxidase activity (measured as DPI-inhibitable NADH
consumption) in lung fibroblasts for control (circles) and
TGF-1-treated (squares) cells. Dose of TGF-
1 was 1
ng/ml. Values are means ± S.D., n =
4.
Certain cytokines have the ability to stimulate the
production of ROS in nonphagocytic
cells(10, 12, 19) . The source(s) of
cytokine-stimulated ROS generation, however, have not been clearly
identified. We have previously reported on the stimulatory effect of
TGF-1 on bovine pulmonary artery endothelial cell
H
O
production without being able to identify
its source(12) . In the present study, we extend this
observation to human lung fibroblasts which, unlike endothelial cells,
demonstrate no release of H
O
extracellularly in
the unstimulated state but, similar to endothelial cells, can be
induced to generate H
O
by TGF-
1. Our study
suggests that the source of this H
O
is the
2-electron reduction of O
via a novel electron transport
system most likely localized in fibroblast plasma membranes. This
electron transport system involves a flavoprotein which specifically
utilizes NADH as the electron donor. Further, the activation of this
flavoprotein oxidase by TGF-
1 appears to require new protein
synthesis.
NADH and NADPH are ubiquitous dinucleotides (each
containing adenine and a nicotinamide ring) that function as carriers
of ``reducing equivalents'' and are used as coenzymes in many
cellular oxidation-reduction reactions. They do this by the transfer of
hydride ion (H, 2 electrons plus a proton) from the
4-position of the nicotinamide ring. The only structural difference
between NADH and NADPH is the presence of an extra phosphate group at
the 2`-position of the adenine nucleotide moiety of NADPH. Although
this extra phosphate group is far from the active redox region (of the
nicotinamide ring) and is of no importance in the hydride ion transfer
reaction, it appears to confer specificity for the enzymes to which
NADPH can bind as a coenzyme.
Based on our findings of NADH-specific
flavoprotein oxidase activity in TGF-1-treated fibroblasts, we can
construct the following simple diagram
The potential for additional ``carriers'' of electron
transport from NADH (donor) to O (acceptor) exists.
Moreover, it cannot be determined conclusively from our current
experiments if the reduction of O
to H
O
involves the intermediate formation of
O
or if this is a direct 2-electron
transfer reaction. We have been unable to demonstrate
O
formation in association with
TGF-
1-induced NADH oxidase activity using superoxide
dismutase-inhibitable ferricytochrome reduction as an assay for
O
release (results not shown). Direct
2-electron reduction of O
has been reported for
flavoprotein oxidases(20) . If we assumed no
``leakage'' of electrons along the electron transport chain
from NADH to O
, the overall stoichiometry of
NADH:H
O
would be expected to be 1:1. A number
of factors may affect the measurement of the concentrations of these
biomolecules under the experimental conditions (using whole cells) of
our study. Therefore, the results of this study cannot be used to
establish stoichiometric relationships.
Several lines of evidence
suggest that the location of the TGF-1-induced NADH oxidase is
most likely at the plasma membrane. Since NADH and NADPH do not cross
plasma membranes, consumption rates of these biomolecules measured in
the extracellular medium are likely to represent oxidation by enzyme(s)
on the outer aspect of the membrane. We considered the possibility of
NADH oxidation by non-flavoprotein oxidases and dehydrogenases or
degradation by nonoxidative pathways and, therefore, specifically
measured DPI-inhibitable consumption of NADH. DPI, previously thought
to be a specific inhibitor of NADPH oxidase, is now recognized as a
relatively nonspecific inhibitor of flavoproteins by direct
binding(17) . It is unlikely that DPI, in the manner used in
our studies, could diffuse through the membrane to inhibit
intracellular flavoprotein oxidoreductases. However, we have assessed
the contributions of intracellular sources of ROS involving
flavoproteins with the use of specific inhibitors of these enzyme
systems, and our results support the likelihood that they are not
involved in TGF-
1-induced H
O
generation.
TGF-1-induced NADH oxidase activity appears to require new
protein synthesis based on the finding of a lag time (>4 h, <8 h)
following exposure to TGF-
1 and its inhibition by cycloheximide
and actinomycin D. It is unlikely that this activity is mediated
directly by TGF-
1 since this peptide has been shown to bind
rapidly to its receptor(s) and is internalized and degraded by
lysosomal enzymes within 4 h at 37 °C(21) . Moreover,
assays for NADH consumption and H
O
release are
made after first washing off the medium and without the reintroduction
of TGF-
1 in assay solutions. Together, these observations suggest
that binding of TGF-
1 to its receptor(s) results in the activation
of a signaling cascade that results in the induction of
H
O
-generating capacity by the cell via an
NADH-specific flavoprotein oxidase. It appears likely that some
component of this protein complex, required for enzymatic activity, has
to be newly synthesized. Our finding of detectable H
O
release in TGF-
1-treated cells in the presence of
cycloheximide and actinomycin D, despite an apparent complete
inhibition of oxidase activity (by the observed lowering of NADH
consumption to control levels, Table 1) suggests that the
relatively smaller amounts of H
O
measured under
these conditions may be unrelated to NADH oxidase activity.
H
O
detected under conditions when protein
synthesis is inhibited might reflect a relative imbalance of oxidant
stress and cellular antioxidant capacity since reduced levels of newly
formed superoxide dismutase, catalase, and components of the
glutathione redox cycle would be expected under these conditions.
Moreover, TGF-
1 alone has been reported to suppress the expression
of these antioxidant enzymes in rat hepatocytes(22) .
Although the source(s) of cytokine-stimulated ROS have remained
largely speculative, some recent studies have attempted to identify the
ROS-generating enzymatic systems. Meier et al.(10) have reported on a superoxide-generating NADPH
oxidase in human skin fibroblasts stimulated by TNF- and IL-1 that
was initially thought to be similar to the NADPH of
neutrophils(10) , but subsequently has been shown to be
structurally and genetically distinct(11) . There are several
important differences between TNF-
- and IL-1-stimulated ROS
production in nonphagocytic cells (10, 19) and the
TGF-
1-induced H
O
production observed in
our studies. First, NADH specificity has not been reported previously.
Secondly, the formation of ROS in response to TNF-
and IL-1 is
immediate, and there is no apparent requirement for new protein
synthesis. Thirdly, we are unable to demonstrate
O
formation in association with
TGF-
1-induced NADH oxidase activity. Finally, we have not observed
similar NADH oxidation or H
O
production in
intact lung fibroblasts in response to TNF-
and IL-1 using short
incubation (immediate activation) or longer exposure (induction of
activity possibly through new protein synthesis, results not shown).
The presence of a growth factor-responsive (epidermal growth factor,
insulin, pituitary extract) NADH oxidase which may function as part of
a redox system at the plasma membrane has been proposed by Brightman
and co-workers(15) . In these studies, NADH oxidase activity
was measured by monitoring NADH utilization and O
consumption, not H
O
production. In fact, ROS
production in association with this activity is apparently lacking. (
)Another recent study has demonstrated the stimulation of
NADH and NADPH oxidase activity in smooth muscle cells by angiotensin
II(23) . NADH oxidoreductase activity has also been shown to be
a major source of O
in unstimulated
bovine coronary artery endothelium(24) . The relationship
between the TGF-
1-induced NADH oxidase activity noted in our
studies and similar activities reported in these former studies is, at
present, unclear.
The best characterized NADPH- or NADH-utilizing
flavoprotein oxidase associated with the formation of ROS is that of
phagocytic cells(25) . Although there are some obvious
similarities such as the involvement of a flavoprotein as part of the
functional enzymatic complex and the use of O as the
electron acceptor, there are also several differences from
TGF-
1-induced NADH oxidase activity. The most striking difference
is the specificity of the oxidase for NADH (and not NADPH). The
kinetics of the formation of ROS by TGF-
1-induced NADH oxidase
activity is characterized by the sustained release of low
concentrations of H
O
for several hours from
fibroblasts in comparison to the rapid, short-lived release of
relatively larger amounts of ROS from the ``respiratory
burst'' of neutrophils. New protein synthesis is not required for
the activation of the NADPH oxidase in neutrophils by the stimulants
studied.
Although the functional significance of TGF-1-induced
NADH oxidase activity remains speculative at this time, there is
increasing recognition of redox-active biomolecules as regulators of
cell function (8, 9) . The kinetics and lower
concentrations of ROS generated by TGF-
1 as well as the lack of
evidence for cell toxicity favor the potential for the
H
O
generated to function as a signaling
molecule. The lack of understanding of post-receptor signaling
mechanisms have led investigators to believe that unidentified pathways
for TGF-
signaling exist(26) . Further studies of the
relationship of TGF-
1-induced NADH oxidase activity and
H
O
production to specific known effects of this
cytokine on target cells may help to uncover redox-sensitive signaling
pathways for the multiple actions of this cytokine.