From the
The cytokine tumor necrosis factor
Tumor necrosis factor
The multitude of
responses elicited by TNF
Emerging evidence has
pointed to the involvement of reactive oxygen species (ROS) as
signaling intermediates for cytokines
(29, 30) . The term
ROS encompasses many species including singlet oxygen, superoxide,
hydrogen peroxide (H
Cytokines such as TNF
We thank A. Bansil for technical assistance with the
FACS analysis, and M. Breitman for the gift of c-fos and
(TNF
) and the
growth factor basic fibroblast growth factor (bFGF) are known to induce
early response genes such as c-fos and c-jun in
various cell types. Activation of AP-1, a heterodimeric complex of Fos
and Jun proteins, is required for matrix metalloproteinase production
and cell proliferation. However, the signaling pathways by which these
two factors influence the expression and activities of AP-1 remain
currently poorly characterized. Several studies have shown that
cytokines induce reactive oxygen species (ROS) production, but growth
factor induction of ROS has not been reported. In the present study we
demonstrate that both TNF
and bFGF induce ROS production, and that
this is a common signaling event involved in the stimulation of
c-fos gene expression in chondrocytes. To our knowledge, this
is the first report directly demonstrating ROS production upon
stimulation with a growth factor. TNF
and bFGF induction of ROS
production is mediated through flavonoid-containing enzymes such as
NADPH oxidase. Moreover, the ROS nitric oxide is not responsible for
the induction of c-fos expression by TNF
and bFGF. In
addition, the inhibitory effects of antioxidants on c-fos expression may account for their protective roles against
proliferative and inflammatory diseases such as cancer, cardiovascular
diseases, and arthritis.
(TNF
)
(
)
is a polypeptide hormone originally identified as a
mediator of hemorrhagic necrosis of tumors in bacillus
Calmette-Guerin-infected mice
(1) . It is synthesized by
different cell types upon stimulation with endotoxin, inflammatory
mediators, or cytokines such as interleukin-1
(2) . TNF
has
been shown to elicit a wide variety of biological effects. It may be
mitogenic, cytostatic, or cytotoxic, depending on the cell type and the
growth state of the
cells
(1, 3, 4, 5, 6) . The
molecular basis for these interesting effects on cell growth and
differentiation is unknown as the intracellular mediators of TNF
action are poorly characterized. Some of the known signal transduction
pathways of TNF
action include coupling to G
proteins
(7, 8) , activation of phospholipase
A
(4, 9, 10, 11) , and
calcium mobilization
(12, 13) .
, however, is not unique to the cytokine
family of regulatory peptides. Many of the cytokine responses such as
mitogenesis and differentiation are also shared by the large family of
growth factors
(14) . One of these, basic fibroblast growth
factor (bFGF), utilizes a receptor with tyrosine kinase activity, a
distinctive feature of the growth factor
family
(15, 16) . bFGF has potent growth and angiogenic
activities
(17) and is produced by various mesodermal and
neuroectodermal tissues, including cartilage, brain, and
retina
(18, 19) . Unlike other growth factors, bFGF does
not possess a signal peptide and thus does not appear to be
secreted
(20) . Although signaling by bFGF may involve the
autophosphorylating tyrosine kinase domain of the receptor which can
interact with phospholipase C-
1
(21) , a G protein-coupled
phospholipase A
activity has also been
reported
(22) . In fact, a mutant bFGF receptor that retains the
tyrosine kinase activity shows diminished ability to stimulate
plasminogen activator production, indicating that the receptor kinase
activity is not sufficient for the full biological activity of
bFGF
(23) . Both TNF
and bFGF are known to induce the
expression of early response gene c-fos in various cell
types
(24, 25) . However, the downstream signaling events
through which TNF
and bFGF elicit the induction of c-fos expression remain to be elucidated. The protooncoproteins Fos and
Jun also referred to as the activator protein-1 (AP-1) complex form
homodimers (e.g. Jun-Jun) or heterodimers (e.g. Fos-Jun) and bind to the AP-1-responsive element in the regulatory
domains of several genes including collagenase, stromelysin,
metallothionein
(26, 27) , and other cellular and viral
genes involved in proliferation
(28) .
O
), nitric oxide (NO), and
hydroxyl radical which are small, diffusible, and ubiquitous molecules,
being produced by virtually every type of cell using diverse enzyme
systems
(31) . Furthermore, it has been demonstrated in several
cell types, such as HeLa cells
(32) and muscle
osteoblasts
(33) , that H
O
can stimulate
c-fos and c-jun gene expression and enhance AP-1
binding activity. Production of ROS upon growth factor activation, on
the other hand, has not been reported. In the present study, we
examined the involvement of ROS in TNF- and bFGF-mediated c-fos induction in a primary culture system of articular chondrocytes.
Reagents
Recombinant human TNF and bFGF
were from Sigma. H
O
was from Fisher Scientific.
N-Acetylcysteine (NAC), ascorbic acid (Asc), and
L-N
-monomethylarginine (L-NMMA)
were also from Sigma. S-Nitroso-N-acetylpenicillamine
(SNAP) was purchased from Biomol Research Laboratories.
Diphenyleneiodonium (DPI) was from Toronto Research Chemicals. Isotope
was from DuPont NEN. Sulfanilamide and naphthylethylenediamine used in
assaying nitrite content were from Sigma.
Cell Culture
Primary cultures of bovine articular
chondrocytes were isolated from bovine articular cartilage as described
in Ref. 34. The cells were plated at 2 10
cells/ml
in 12 ml of Ham's F-12 media containing 3% antibiotics and 5%
fetal bovine serum. The cells were allowed to recover for 24 h at 37
°C in a humidified atmosphere supplemented with 5% CO
.
Northern Blot Analysis
Total RNA was isolated by
the acidified guanidine isothiocyanate method
(35) and subjected
to electrophoresis on a denaturing gel. Denatured RNA samples (12
µg) were analyzed by gel electrophoresis in a denaturing 1% agarose
gel, transferred to a nylon membrane (Bio-Rad), cross-linked with an
ultraviolet cross-linker (Stratagene UV Stratalinker 1800), and
hybridized with P-labeled rat c-fos cDNA. The
blots were subsequently stripped and reprobed with
P-labeled rat
tubulin cDNA.
Fluorescence-activated Cell Sorting (FACS)
Analysis
Chondrocytes were treated with TNF (30 ng/ml) or
bFGF (10 ng/ml) for 4 h in the presence of dihydrorhodamine 123 (DHR)
(2 µM) (Molecular Probes) with or without DPI (2
µM). Both DHR and DPI were dissolved in dimethyl
sulfoxide. During the cellular production of ROS, the intracellular DHR
was irreversibly converted to the green fluorescent compound rhodamine
123 (R123) (500-540 nm). R123 was membrane-impermeable and
accumulated in the cells. Chondrocytes were fixed for 20 min in 1.5%
paraformaldehyde (Sigma) and the cellular R123 fluorescence intensity
of 5000 chondrocytes was measured for each sample by flow cytometry
using a fluorescein isothiocyanate argon laser with the excitation
source at 488 nm (Coulter Epics C flow cytometer) (36).
Measurement of NO Production
NO production was
measured as the amount of nitrite, the stable end product of NO,
released into the culture supernatant. Nitrite concentration was
determined in cell-free culture supernatants using the
spectrophotometric method based on the Griess reaction
(37) .
Briefly, samples were reacted in equal volume with 1% sulfanilamide,
0.1% naphthylethylenediamine (Sigma), and 5% phosphoric acid at room
temperature for 10 min. The nitrite concentration was determined by
absorbance at 540 nm in comparison with standard solutions of sodium
nitrite prepared in the same medium.
RESULTS AND DISCUSSION
In the current study, we examined the effects of TNF and
bFGF on ROS production and its relationship with c-fos expression in chondrocytes. Three criteria were utilized to assess
the relevance of ROS as putative second messengers in the induction of
c-fos by the two factors: (i) addition of ROS should mimic the
inducers' biological effects, (ii) decreasing ROS production or
inactivating ROS should inhibit the induction of c-fos expression by TNF
and bFGF, and finally (iii) TNF
and
bFGF should stimulate ROS production. Hydrogen peroxide, a
membrane-permeable reagent physiologically produced in large amounts by
granulocytes and macrophages during inflammatory
processes
(31, 38, 39) , has been widely used to
assess the effects of ROS. H
O
is capable of
inducing c-fos(32, 33) and DNA synthesis
(40) and activating the transcription factor NF
B in various
systems
(41) . We therefore first examined the effect of
H
O
on c-fos mRNA expression in primary
cultures of bovine articular chondrocytes. Cells were treated with 100
µM hydrogen peroxide for various time points, and
c-fos mRNA levels were determined by Northern blot analysis.
As shown in Fig. 1A, we found that H
O
increased c-fos mRNA levels in chondrocytes optimally at
30 min and levels decreased to base-line levels by 2 h. These results
are consistent with reactive oxygen species involvement in c-fos induction. Similar transient increases in c-fos mRNA
levels were observed with TNF
and bFGF (data not shown).
Figure 1:
Effects of HO
and antioxidants on c-fos expression. A, effect of
H
O
on c-fos mRNA levels. Chondrocyte
cultures were stimulated with H
O
(100
µM) at different time points as indicated. B, the
antioxidants NAC and Asc inhibit TNF
- and bFGF-induced c-fos mRNA levels. Chondrocyte cultures were preincubated with NAC (30
mM) or Asc (100 µM) for 2 h before the addition
of bFGF (10 ng/ml) or TNF
(30 ng/ml) for 30 min. Both human
recombinant TNF
and bFGF were dissolved in phosphate-buffered
saline with 0.1% bovine serum albumin. NAC and Asc were first dissolved
in Ham's F-12 medium containing 5% (v/v) fetal bovine serum, then
neutralized with sodium hydroxide. Total RNA from bovine articular
chondrocytes was isolated, and the c-fos mRNA levels were
determined by Northern blot analysis as described under
``Materials and Methods.'' The blots were subsequently
stripped of DNA and reprobed with
P-labeled rat
tubulin cDNA.
We
next determined the effects of two antioxidants, NAC and Asc, on the
induction of c-fos expression. NAC and Asc are effective free
radical scavengers; the former is known to increase intracellular
glutathione levels, which in turn control the concentration of ROS
within cells via glutathione peroxidase
(31) . On the other hand,
Asc itself is highly reactive toward radicals and proven to be a
versatile scavenger
(42) . Fig. 1B demonstrates
that TNF (30 ng/ml) and bFGF (10 ng/ml) increased c-fos mRNA levels in chondrocytes. However, the addition of antioxidants
NAC (30 mM) and Asc (100 µM) 2 h prior to
stimulation with TNF
and bFGF attenuated the induction of
c-fos mRNA levels. NAC was more effective than Asc in reducing
the induction of c-fos mRNA expression. Both antioxidants did
not affect the mRNA levels of the housekeeping gene tubulin. Taken
together, these results suggest that decreasing ROS levels by
antioxidants suppresses TNF
and bFGF induction of c-fos expression in chondrocytes.
and
interleukin 1 have been shown to stimulate H
O
production by fibroblasts and chondrocytes (29, 30); however, the
effects of growth factors on ROS production in cells have not been
reported. To directly determine whether TNF
and bFGF induce ROS
production, chondrocyte cultures loaded with DHR (2 µM)
were stimulated with TNF
or bFGF and then examined by FACS
analysis. Although c-fos expression was induced at 30 min, ROS
production induced by either TNF
and bFGF or the ROS
H
O
itself could not be detected by FACS
analysis at this time point (data not shown), probably due to
experimental limitations of the assay. However, both TNF
and bFGF
stimulated ROS production in chondrocytes after a 4-h incubation as
shown by a shift in the logarithmic fluorescence intensity to the right
(Fig. 2, d and f) as compared to constitutive
ROS production (Fig. 2c). These data provide direct
evidence of ROS production by chondrocytes after stimulation with both
TNF
and bFGF. One class of enzymes that are known to give rise to
various types of ROS is the flavonoid-containing enzymes. Therefore, we
examined the effects of DPI, a potent inhibitor of flavonoid-containing
enzymes such as NADPH oxidase and nitric oxide synthase
(NOS)
(43) , on TNF
and bFGF induction of ROS production.
Pretreatment of chondrocytes with DPI (2 µM) completely
abolished the induction of ROS production as demonstrated by a backward
shift of green fluorescence to that of basal levels (Fig. 2,
e and g). The inhibition by DPI of both TNF
- and
bFGF-induced ROS production indicates a role for flavonoid-containing
enzymes in the induction process. Although further research is
necessary to elucidate the mechanisms by which these factors stimulate
ROS production, it is possible that increases in arachidonic acid or
its metabolites in response to cytokines and growth
factors
(22, 44, 45) may serve as intermediates
in the activation of enzymes such as NADPH
oxidase
(46, 47) .
Figure 2:
DPI inhibits TNF- and bFGF-induced
ROS production in chondrocytes. With time, DHR by itself caused a shift
in fluorescence to the right as shown in panels a-c. A
dottedline was drawn through the mean fluorescence
intensity of the control (panel c) with DHR alone for 4 h.
After incubation with TNF
or bFGF for 4 h in the presence of DHR,
there was an additional shift in logarithmic fluorescence intensity as
indicated in panels d and f. In panels e and
g, DPI abolished the fluorescent shift stimulated by TNF
and bFGF, respectively.
Next, the effects of DPI on
TNF- and bFGF-induced c-fos mRNA levels were examined by
Northern blot analysis. Pretreatment of chondrocytes with DPI
significantly decreased TNF
and bFGF induction of c-fos mRNA levels (Fig. 3). Under identical conditions, tubulin
mRNA levels were not altered by DPI. These data indicate that
flavonoid-containing enzymes are involved in the regulation of
c-fos expression mediated by TNF
and bFGF in
chondrocytes. The concomitant inhibitory effects of DPI on ROS release
and on induction of c-fos support the involvement of ROS as a
shared mechanism by which TNF
and bFGF induce c-fos expression.
Figure 3:
Diphenyleneiodonium also inhibits the
induction of c-fos expression by TNF and bFGF.
Chondrocyte cultures were pretreated with DPI (2 µM) for
30 min before the addition of TNF
(30 ng/ml) or bFGF (10 ng/ml)
for 30 min. Measurements of c-fos and tubulin mRNA levels were
as described under Fig. 1.
TNF has been shown to stimulate nitric oxide
(NO) production in a variety of cell types
(48, 49) . The
free radical NO has received increasing recognition as an important
physiologic messenger molecule with regulatory roles in the nervous,
immune, and cardiovascular systems
(50) . Thus, we investigated
the capacity of bovine chondrocytes to produce NO and the potential
influences of NO, if any, on c-fos expression (Fig. 4).
TNF
was found to increase NO production by 11-fold when compared
with that of the control (Fig. 4A). As expected, the NOS
inhibitor L-NMMA blocked the stimulation of NO production by
75% (Fig. 4A). In contrast, bFGF did not induce NO
production in chondrocytes; thus, NO is not involved in bFGF induction
of c-fos expression. Since TNF
increased NO production,
we examined the effect of L-NMMA on TNF
-induced c-fos mRNA levels (Fig. 4B). The data demonstrated that
L-NMMA did not alter constitutive or TNF
- or bFGF-induced
c-fos mRNA expression. In summary, NO does not appear to be
involved in the induction of c-fos expression by cytokines or
growth factors. Furthermore, the inability of increased levels of
extracellular NO using SNAP to induce c-fos expression
provides further evidence that NO production is not involved in the
regulation of c-fos mRNA expression in chondrocytes. However,
we could not exclude the possibility that NO released as the result of
TNF
stimulation may participate in other signaling processes
important in regulating the expression of other genes. Moreover,
abnormal levels of NO production in response to heightened cytokine
secretion may also result in cytotoxicity and tissue damage, a process
implicated in some pathological situations
(50, 51) .
Figure 4:
Role of NO in regulation of c-fos expression. A, effects of TNF and bFGF on nitric
oxide production. Chondrocytes were first treated with 250
µML-NMMA 2 h prior to the addition of either
TNF
(30 ng/ml) or bFGF (10 ng/ml). After 72 h of incubation,
nitrite content was determined as described under ``Materials and
Methods.'' Values shown are means (± S.E.) of three
independent experiments, each done in triplicate. B,
L-NMMA has no effect on TNF
- and bFGF-induced c-fos mRNA levels. Chondrocyte cultures were first incubated with
L-NMMA (250 µM) for 2 h before adding TNF
(30 ng/ml) or bFGF (10 ng/ml). After 30 min, total RNA was isolated and
c-fos mRNA levels were determined as described in Fig. 1.
C, the organic NO donor, SNAP, cannot induce c-fos mRNA levels. Chondrocytes were stimulated with 100 µM
SNAP for the time periods as indicated. H
O
(lane 2) was used as a positive control in this
experiment.
Although cytokine and growth factor stimulation of c-fos transcription is thought to be mediated by mitogen-activated
protein (MAP) kinase phosphorylation of the transcription factor
ELK-1/TCF (52-54), a second messenger common to both factors in
this early gene response has not been identified. In our study, we
demonstrated that cytokine and growth factor stimulation of ROS
production by flavonoid-containing enzymes is a common signaling
mechanism involved in the induction of c-fos expression in
chondrocytes. More important, this is the first report directly
demonstrating ROS production upon stimulation with a growth factor.
Recently, it was demonstrated that ROS produced by HO
or ionizing irradiation are capable of stimulating MAP
kinases
(55, 56) . Therefore, it is conceivable that ROS
induction of c-fos mRNA levels occurs through activation of
MAP kinases, which phosphorylate and thus activate transcription
factors such as ELK-1/TCF, which in turn regulate c-fos promoter activity (52-54). Although ROS production may be a
component of normal signal transduction in many cell types, it is
likely that abnormal production of ROS stimulated by elevated levels of
cytokines and growth factors may inappropriately activate early
response genes such as c-fos and c-jun leading to the
overexpression of metalloproteinases and uncontrolled cell
proliferation. The inhibition of TNF
- and bFGF-induced c-fos mRNA levels by antioxidants also led us to hypothesize that
antioxidants may prove useful to impede disease progress by
down-regulating AP-1 and their responsive genes such as
metalloproteinases. Hence, antioxidant inhibition of c-fos expression may serve to explain, at least in part, the ability of
antioxidant mixtures to improve health conditions in patients with
diabetes, arthritis, hypertension, and other age-related
diseases
(57) .
, tumor necrosis factor
; bFGF,
basic fibroblast growth factor; AP-1, activator protein-1; ROS,
reactive oxygen species; NAC, N-acetylcysteine; Asc, ascorbic
acid; L-NMMA,
L-N
-monomethylarginine; SNAP,
S-nitroso-N-acetylpenicillamine; DPI,
diphenyleneiodonium; FACS, fluorescence-activated cell sorting; DHR,
dihydrorhodamine 123; R123, rhodamine 123; NOS, nitric oxide synthase;
MAP, mitogen-activated protein.
-tubulin cDNAs.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.