(Received for publication, November 27, 1995)
From the
Tumor necrosis factor (TNF) activates both p42 and p44
mitogen-activated protein kinases (MAPK) in human FS-4 fibroblasts,
cells for which TNF is mitogenic. We now show that TNF activates p42
MAPK in two cell lines whose growth is inhibited by TNF. A mutant TNF
that binds only to the p55 TNF receptor (TNFR) produced a similar
degree of activation as wild-type TNF in FS-4 fibroblasts, indicating
that the p55 TNFR is sufficient to mediate p42/p44 MAPK activation. The
upstream intracellular signals that couple the TNFR to MAPK activation
are still poorly defined. We now show that neither phorbol
ester-sensitive protein kinase C nor G link TNF to
p42/p44 MAPK activation, because pretreatment of FS-4 cells with
phorbol ester to down-regulate protein kinase C or pretreatment with
pertussis toxin to block G
does not inhibit p42/p44
MAPK activation by TNF. To further analyze MAPK activation in FS-4
cells, we compared p42/p44 MAPK activation by TNF and epidermal growth
factor (EGF). While tyrosine phosphorylation of p42/p44 MAPK was
detected almost immediately (30 s) after stimulating cells with EGF,
TNF-induced tyrosine phosphorylation was detected only after a more
prolonged time interval (initially detected at 5 min and peaking at
15-30 min). In addition, the anti-inflammatory drug sodium
salicylate, previously demonstrated to inhibit NF-
B activation by
TNF, blocked the activation of p42/p44 MAPK in response to TNF but not
in response to EGF. These findings demonstrate that the TNF and EGF
receptors utilize distinct signaling molecules to couple to MAPK
activation. Elucidation of the mechanism whereby sodium salicylate
blocks TNF-induced p42/p44 MAPK activation may help to clarify
TNF-activated signaling pathways.
Tumor necrosis factor (TNF), ()a cytokine originally
described as a mediator of endotoxin-induced hemorrhagic necrosis of
tumors, possesses potent immunomodulatory capacities and is believed to
play key roles in inflammation, septic shock, and
cachexia(1, 2) . Two TNF receptors (TNFRs) of 55 kDa
(p55) and 75 kDa (p75) are expressed on many types of cells and
transduce the TNF signal(3, 4) . The extracellular
portions of the receptors possess structural features also present in
the extracellular domain of the nerve growth factor receptor and other
receptors comprising the TNFR superfamily(3) . Although the
intracellular domains of the p55 and the p75 TNFRs do not display
significant homology to each other, the cytoplasmic portion of the p55
TNFR contains a death domain also found in the Fas antigen, necessary
for signaling cell death(5) . Recent studies have identified
several proteins associated with the cytoplasmic domains of the p55 (6, 7, 8) and p75 (9) TNFRs.
The
initial event in TNF signal transduction involves association of a
trimeric TNF molecule with its receptor and subsequent receptor
oligomerization. There is evidence for the involvement of several
receptor-distal elements in the propagation of the TNF signal,
including GTP-binding proteins(10, 11) , protein
kinase A(12) , and protein kinase C
(PKC)(13, 14) . In addition, TNF has been shown to
activate phosphatidylcholine-specific phospholipase C (15) and
cytosolic phospholipase A(16) and to cause an
increase in arachidonic acid
metabolism(16, 17, 18) . There is also
evidence for sphingomyelinase activation by TNF, leading to the
generation of ceramide, a potent second messenger(19) .
Finally, TNF has been shown to activate several members of a large and
growing family of mitogen-activated protein kinases
(MAPKs)(20, 21, 22, 23, 24) .
MAPKs constitute a family of related and evolutionarily conserved
serine/threonine kinases that are important in cell growth and
differentiation(25, 26, 27) . They become
activated by phosphorylation on threonine and tyrosine in response to
many external stimuli. MAPKs preferentially recognize a minimal
substrate consensus sequence, Ser/Thr-Pro(28) , present in many
proteins. Accordingly, MAPKs appear to have many possible substrates,
including transcription factors such as STAT1 (29) and the
serum response factor accessory protein Elk-1 (30) , as well as
cytosolic proteins such as cytosolic phospholipase
A
(31) . Consistent with the role of MAPKs in
modulating transcription factor function is the observation that MAPKs
frequently translocate from the cytoplasm into the nucleus following
growth factor stimulation(32) . Three MAPK subfamilies have
been identified, consisting of the p42 and p44 MAPKs (also termed
extracellular signal-regulated kinases (ERKs)), the c-Jun N-terminal
kinases (JNK)/stress-activated protein kinases (SAPK), and the p38
MAPKs (20, 33, 34, 35) (see (36) for a review). Although each of the subfamilies appears to
have its own set of activators, there is a certain amount of crossover
in the pathways. For example, while MAPK-ERK kinase kinase (MEKK)
preferentially functions in the JNK/SAPK pathway, it can also activate
MAPK-ERK kinase (MEK), located in the p42/p44 MAPK pathway (37, 38) . TNF can activate all three MAPK
subfamilies(20, 21, 22) . In particular, TNF
has been shown to activate both MEK (39, 40) and MEKK (40) . The activation pathway for p42/p44 MAPK by receptor
tyrosine kinases typically involves Ras, either Raf or MEKK, and
MEK(41, 42, 43, 44) . While there is
evidence for both MEK (39, 40) and MEKK (40) involvement in TNF actions in some cells, it is not clear
what other components may participate in the TNF-mediated activation of
p42/p44 MAPK.
In earlier work, we showed that TNF activated p42/p44 MAPK in normal human FS-4 fibroblasts(21) . The goal of this study was to further characterize the activation of p42/p44 MAPK by TNF and to compare this activation with that mediated by other agents, especially EGF. Our findings indicate that in addition to activating p42/p44 MAPK with different kinetics, TNF and EGF-activated pathways differ in their susceptibility to inhibition by sodium salicylate. Whereas TNF-induced p42/p44 MAPK activation was strongly inhibited, sodium salicylate showed no significant effect on MAPK activation by EGF. It is possible that this newly demonstrated inhibition of TNF signaling contributes to the anti-inflammatory action of sodium salicylate. Elucidation of the mechanism of the inhibitory effect of sodium salicylate may help to clarify the signaling pathways activated by TNF.
Figure 1:
Activation of p42 MAPK and p44 MAPK by
TNF in various cell lines. A, serum-starved FS-4 cells were
treated for 15 min with 20 ng/ml TNF, 1 ng/ml IL-1, 50 µg/ml
poly(I)poly(C), or left untreated (Ctrl). HeLa and HT-29
cells were each stimulated for 15 min with 20 ng/ml TNF or left
untreated (Ctrl). The cells were then lysed as described under
``Experimental Procedures.'' Following SDS-polyacrylamide gel
electrophoresis, proteins were transferred to an Immobilon-P membrane
and probed with 1B3B9, a mouse monoclonal antibody to p42 MAPK.
Immunocomplexes were detected using a biotinylated secondary antibody
and streptavidin-alkaline phosphatase complex. Arrows indicate
the shift from a faster migrating hypophosphorylated form of p42 MAPK (p42) to a more slowly migrating phosphorylated form of p42
MAPK (pp42). B, the same lysates from FS-4, HeLa, and
HT-29 cells used for blotting in A were subjected to an in-gel
kinase assay utilizing MBP, as described under ``Experimental
Procedures.'' The arrow indicates the position of
activated pp42 MAPK. Locations of molecular mass standards are shown at
the left in kilodaltons. C, serum-starved FS-4 cells were
stimulated with 20 ng/ml TNF or mutant
(Trp
/Thr
) TNF (mutTNF) for 15 min or
left untreated (Ctrl) and then lysed. Lysates were
subsequently Western blotted and probed with anti-Tyr(P) .
Immunocomplexes were detected with the aid of Protein A and
chemiluminescence. Arrows indicate positions of activated (pp42 and pp44) MAPKs. dsRNA,
double-stranded RNA.
Figure 2: Kinetics of TNF- and EGF-induced activation of p42/p44 MAPK in FS-4 cells. Serum-starved FS-4 cells were either left untreated or treated with TNF (20 ng/ml) or EGF (30 ng/ml) for the indicated times and then lysed. The lysates were Western blotted and probed with anti-Tyr(P) (A (top panel) and B). The same lysates were also probed with anti-p42 MAPK (A, bottom panel). Immunocomplexes were detected with Protein A and chemiluminescence, as described in the legend to Fig. 1C. Arrows indicate positions of activated MAPKs (pp42 and pp44). Locations of molecular mass markers are shown at the left in kilodaltons.
Figure 3:
Effect of pertussis toxin pretreatment on
TNF-induced tyrosine phosphorylation of p42/p44 MAPK. Serum-starved
FS-4 cells were either left untreated or treated for 18 h with 500
ng/ml of PT before stimulation for 15 min with TNF (20 ng/ml), 10 min
with EGF (30 ng/ml), or 10 min with LPA (100 ng/ml). As a control,
cells were also treated with dimethyl sulfoxide alone (used as a
carrier for LPA), which did not activate p42/p44 MAPK (data not shown).
Lysates were then generated, Western blotted, and probed with
anti-Tyr(P) (top panel, -PTyr) and anti-p42 MAPK (bottom panel). Arrows denote positions of activated
MAPKs (pp42 and pp44).
Figure 4:
Effect of pretreatment with a high dose of
phorbol ester on activation of p42/p44 MAPK by TPA, TNF, or EGF.
Serum-starved FS-4 cells were either left untreated or treated for 24 h
with 1 µg/ml TPA before stimulation for either 15 min with TNF (20
ng/ml) (lanes 3 and 7), 10 min with EGF (30 ng/ml) (lanes 4 and 8), or 10 min with TPA (20 ng/ml) (lanes 2 and 6). Lysates were then generated, Western
blotted, and probed with anti-Tyr(P) (top panel, -PTyr) and with anti-p42 MAPK (bottom panel). Arrows denote positions of activated (pp42 and pp44) MAPKs.
Figure 5:
Effects of sodium salicylate pretreatment
on TNF-induced tyrosine phosphorylation of p42/p44 MAPK. A,
serum-starved FS-4 cells were treated for the indicated times with 20
mM sodium salicylate (NaSal). They were then either
left untreated or stimulated for 15 min with 20 ng/ml TNF or mutant
(Trp/Thr
) TNF (mutTNF). Cells were
then lysed, and lysates were Western blotted and probed with
anti-Tyr(P) (
-PTyr). B, serum-starved FS-4 cells
were either left untreated or treated for 1 h with 20 mM sodium salicylate. They were then left unstimulated (lanes 1 and 5), stimulated for 15 min with 20 ng/ml TNF (lanes 2 and 6), stimulated for 10 min with 30 ng/ml
EGF (lanes 3 and 7), or co-stimulated for 15 min with
both TNF and EGF at the above concentrations (lanes 4 and 8). Lysates were then generated, blotted, and probed with
anti-Tyr(P). Arrows in both panels A and B denote positions of activated (pp42 and pp44)
MAPKs.
Previous studies have demonstrated the TNF-mediated
activation of p42/p44 MAPK in several different types of
cells(20, 21, 22, 23, 24) .
However, the signaling pathways linking the TNFRs to p42/p44 MAPK
activation remain largely unknown. The main goal of the present study
was to compare p42/p44 MAPK activation by TNF and by EGF in the human
diploid FS-4 fibroblasts and to determine whether the pathways of
activation by these agents can be distinguished. A clear difference was
seen in the kinetic patterns of activation by the two agents, as judged
by the increases in phosphotyrosine contents of p42 and p44 MAPK, such
that EGF produced both a more rapid and a more sustained activation
than TNF. In view of an earlier observation that TNF-induced NF-B
activation can be inhibited by acetylsalicylic acid or sodium
salicylate(56) , we examined the effect of the latter agent on
p42/p44 MAPK activation in our system. Sodium salicylate produced a
marked inhibition of TNF-induced p42 and p44 MAPK tyrosine
phosphorylation but failed to suppress EGF-induced activation. This
different response to sodium salicylate indicates that TNF and EGF
produce p42/p44 MAPK activation via different routes and suggests an
important role for phospholipid metabolism in the TNF-activated
pathway. Our demonstration of the inhibitory action of sodium
salicylate on TNF-induced p42/p44 MAPK activation may help to design
new approaches to the analysis of TNF signaling.
Our finding that
the p55 TNFR is sufficient for activation of p42/p44 MAPK confirms
similar findings by others in macrophages(57) , and complements
earlier observations that the p55 receptor independently can mediate
TNF's antiviral activity (58) and also stimulate PKC,
NF-B, phospholipase A
, and sphingomyelinase activities (59) . The cytoplasmic portion of the p55 receptor has been
shown to possess a so-called ``death domain'' responsible for
signaling cytotoxicity, which is homologous to the intracellular
portion of the Fas antigen(5) . Since Fas has also been shown
to activate MAPK(60) , it will be of interest to determine
whether it is the death domain that mediates the activation of p42/p44
MAPK by the p55 receptor. Pagès et al.(25) have demonstrated that p42/p44 MAPK is required for
the proliferation of fibroblasts in response to growth factor
stimulation. We showed that TNF activates p42/p44 MAPK in FS-4
fibroblasts, cells for which it is mitogenic(45) . However, we
now show that TNF also activates p42 MAPK in HeLa and HT-29 cells, in
which TNF is cytostatic(46) . These findings indicate that
while p42 MAPK activation may be required for growth factor-induced
mitogenesis, it probably serves a more general role in TNF signaling.
The kinetics of p42/p44 MAPK tyrosine phosphorylation in FS-4 fibroblasts in response to TNF treatment, with increased phosphorylation beginning at 5 min and ending after approximately 40 min of stimulation, are in agreement with the findings of other investigators in different cell types(24, 57) . This relatively transient TNF-induced tyrosine phosphorylation of p42/p44 MAPK is also consistent with the findings demonstrating that while TNF is a potent activator of the SAPK/JNK pathway, it is a relatively weak activator of p42/p44 MAPK(33, 61) . The relatively brief period of p42/p44 MAPK activation by TNF and the apparent existence of redundancy in target selection by members of the MAPK subfamilies raise questions as to the possible functions of p42/p44 MAPK in TNF signaling. For example, the Elk-1 transcription factor, known to be phosphorylated by p42/p44 MAPK in response to TPA treatment, appears to be principally phosphorylated by JNK in response to IL-1 (and most probably TNF) stimulation(62) . Further studies will be required to determine the specific downstream targets of TNF-activated p42/p44 MAPK.
Our finding that sodium salicylate
blocks the TNF-mediated tyrosine phosphorylation of p42/p44 MAPK in
FS-4 fibroblasts complements earlier observations demonstrating the
sodium salicylate-mediated inhibition of NF-B activation in
response to TNF and other agents(56) . In addition to its
effects on NF-
B, sodium salicylate has been shown to induce DNA
binding of the heat shock transcription factor in HeLa cells (63) and to function as a signaling molecule in the response of
plants to infection with various pathogens(64, 65) .
Recent work has also demonstrated that sodium salicylate, at
suprapharmacological concentrations, can inhibit inducible nitric-oxide
synthase expression and nitrite production in murine
macrophages(66) . To our knowledge, the data described in our
present paper represent the first demonstration of sodium
salicylate's interference in a cytokine-induced kinase cascade.
Along with the above mentioned actions of sodium salicylate, this
interference with the TNF/MAPK pathway may contribute to the well
documented anti-inflammatory effect of sodium salicylate in
vivo. It is likely that sodium salicylate may also block the
pathways for MAPK activation by other inflammatory cytokines and/or
that sodium salicylate may block other signaling pathways employed by
TNF and other inflammatory agents.
The sodium salicylate-mediated
inhibition of TNF-induced p42/p44 MAPK activation was clearly
demonstrable only at the relatively high sodium salicylate dose of 20
mM. This same dose has also been used by others to demonstrate
several other actions of sodium salicylate (63, 66) .
In view of the pH dependence of sodium salicylate action in at least
one system, in which decreased local pH increased the effectiveness of
sodium salicylate(63) , it is possible that lower
concentrations of sodium salicylate will inhibit the TNF-induced
p42/p44 MAPK pathway under some conditions. It will be important to
determine the specific step at which sodium salicylate interferes with
the activation of p42/p44 MAPK by TNF. In view of its inhibitory action
on NF-B activation by TNF(56) , it is possible that sodium
salicylate exerts a global inhibitory effect on TNF signaling, and acts
at a far upstream, TNF receptor-proximal site. In this regard, our
preliminary experiments in FS-4 cells indicate that the TNF-mediated
activation of JNKs is also significantly decreased by sodium salicylate
pretreatment. A principal mechanism of action of nonsteroidal
anti-inflammatory drugs is thought to be interference with pathways of
arachidonic acid metabolism(67, 68, 69) .
Because TNF has been demonstrated to cause an increase in arachidonic
acid metabolism (17, 18) and because arachidonic acid
itself can cause MAPK activation in at least one cell
type(70) , sodium salicylate may be blocking the TNF-mediated
induction of p42/p44 MAPK by interfering with arachidonic acid
metabolism. Alternatively, because sodium salicylate has been shown to
inhibit phospholipase C activity (71) and because TNF is known
to activate phospholipase C-dependent pathways(15) , sodium
salicylate may be blocking the TNF-mediated induction of p42/p44 MAPK
by interfering with phospholipase C metabolism. In any case, our
present work broadens the spectrum of potential targets for the
anti-inflammatory actions of sodium salicylate and could serve as a
tool to further dissect TNF signaling pathways.