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,
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Ludwig Institute for Cancer Research, London, United Kingdom;
Laboratory for Experimental Internal Medicine, Academic Medical Centre, Amsterdam, The Netherlands;
Laboratory for Molecular Biology, Flemish Institute for Biotechnology, Gent, Belgium;
§
The Randall Institute, Kings College, London, United Kingdom; and
¶
Department of Biochemistry and Molecular Biology, University College of London, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) migration, but the
signaling pathways mediating this response have not been established.
Here we report that stimulation of the 55-kDa TNF receptor (TNFR-1)
induced an overall decrease in filamentous actin (F-actin), inhibited
CSF-1- and Cdc42-dependent filopodium formation, and stimulated
macropinocytosis. Using a panel of TNFR-1 mutants, the regions of the
receptor required for each of these responses were mapped. The decrease
in F-actin required both the death domain and the membrane proximal
part of the receptor, whereas inhibition of filopodium formation and
increased pinocytosis were only dependent upon a functional death
domain. When the TNF-induced decrease in F-actin was inhibited using
either receptor mutants or the compound D609, TNF-stimulated actin
reorganization at the cell cortex became apparent. This activity was
dependent upon the FAN-binding region of TNFR-1. We conclude that
different domains of TNFR-1 mediate distinct changes in the M
cytoskeleton, and that the ability of TNF to inhibit M chemotaxis
may be due to decreased filopodium formation downstream of
Cdc42. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)2 in particular
function as important mediators of TNF in pathophysiology. In these
cells, TNF triggers the synthesis and secretion of many proinflammatory
cytokines, including TNF itself, as well as lipid mediators such as
platelet-activating-factor and prostaglandins (2, 3). M activation
is itself dependent upon stimulation by the membrane-bound form of TNF
expressed by CD4+ Th1 cells (4, 5), which promotes
important M effector functions such as the production of oxygen
radicals and nitric oxide, the synthesis of antibiotic enzymes, and the
fusion of phagosomes with lysosomes. Thus, TNF-induced activation is
essential for the M-mediated destruction of pathogens and tumor
cells (6). In addition, TNF has profound effects on phagocyte
locomotion, inhibiting the migratory response of M toward
chemotactic stimuli, an effect that probably serves to retain M at
the actual site of infection. This migration-inhibitory effect,
which is also observed in TNF-stimulated neutrophils, seems to be
mediated by an TNF-induced loss of chemotactic responses (7, 8, 9, 10).
Indeed, we recently showed that TNF abrogated M chemotaxis toward
CSF-1 but had no effect on CSF-1-stimulated chemokinesis (11).
Therefore, TNF action in M includes specific effects on M
locomotion.
TNF acts by stimulating two different types of plasma
membrane-localized receptors: TNF receptor 1 (TNFR-1) and TNF receptor
2 (TNFR-2), both of which are expressed on M. Although these TNFRs
are independently active in TNF signaling (12), the large majority of
TNF-induced signaling events can be mediated solely by TNFR-1 (reviewed
in 13 . The addition of TNF to TNFR-1 results in receptor
aggregation. Subsequently, a variety of adapter molecules is recruited
to the intracellular part of the receptor, different domains apparently
interacting with specific adapter molecules, which in turn activate
distinct signal transduction pathways. Among the receptor
associating-proteins identified is a protein called FAN, which binds
the TNFR at amino acids 307–321 and activates neutral sphingomyelinase
activity (14). Directly adjacent to the FAN-binding part of the
receptor is the death domain (amino acids 326–413), which can bind the
TNFR-1-associated death domain protein (15). The latter protein can
subsequently recruit other signaling proteins, including the
TNFR-associated proteins 1 and 2 (TNFR-associated factor-1 and
TNFR-associated factor-2) (16, 17), the Fas-associated protein
with death domain (17), receptor-interacting protein (18), and others.
The protein complex thus formed mediates a number of responses,
including TNF-induced activation of NF-
B, c-Jun N-terminal kinase
(stress-activated protein kinase), caspases, and apoptosis (19).
Finally, the membrane proximal region of the receptor (amino acids
205–258) binds and, upon receptor activation, stimulates a
phosphatidylinositol-4-phosphate 5-kinase (20). In addition, this part
of the receptor together with the death domain induces nitric oxide
synthase (21).
The role of the different TNFR-1 domains and associated signal
transduction pathways in mediating the effects of TNF on M migration
has not been investigated. However, a group of proteins known as the
Rho family of small GTPases has been implicated in regulating cell
migration. Members of this family, including Cdc42, Rac, and Rho, play
a major role in remodeling the actin cytoskeleton in response to
external stimuli (reviewed in Refs. 22 and 23). Cdc42 specifically
induces the formation of filopodia in Swiss 3T3 cells (24, 25), whereas
Rac and Rho regulate lamellipodium and stress fiber formation,
respectively (26, 27). Recently, we demonstrated that Rho family
members have similar activities in M: Cdc42 mediates filopodium
formation, whereas Rac regulates lamellipodium production (28). As
changes in cell locomotion are dependent upon controlled remodeling of
the actin cytoskeleton, it is generally assumed that Rho family members
have an important function in directing cell motility (29). Therefore,
the TNF-induced inhibition of M chemotaxis might well be brought
about by interference with either the activation of Rho family proteins
or downstream signal transduction to the actin cytoskeleton.
Little is known of the signaling pathways regulating TNF-induced actin
reorganization in any cell type; in particular, the effects of TNF on
the actin cytoskeleton of M have remained unexplored. To obtain more
insight into the molecular mechanisms by which TNF controls M
migration, we have investigated the effects of TNF on the M actin
cytoskeleton and on the actin reorganization induced by CSF-1 and Rho
family proteins. We have subsequently used TNFR-1 mutants to determine
which portions of the receptor are responsible for signaling to the
actin cytoskeleton in M.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P388D1 cells, 4-4 cells, and J774 cells were cultured in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) with 10% FCS according to routine procedures. For microinjection and cell-stimulation experiments, cells were seeded on 13-mm coverslips, switched to RPMI 1640 without FCS after 24 h, and microinjected 24 h later. In cell-stimulation experiments, cells were washed with RPMI 1640 without FCS at 24 h after seeding and maintained without FCS for 48 h. Experiments were performed in the same medium. Bac1.2F5 cells were cultured and passaged as described previously (11). For experiments (unless otherwise indicated), cells were starved overnight in RPMI 1640 without serum. Experiments were performed in the same medium.
Construction of hTNF-R55 mutants
The cDNA of human TNF-R55 (generously provided by Drs.
W. Lesslauer and H. Loetscher, Hoffmann-La Roche, Basel,
Switzerland) was cloned into the eukaryotic expression vector pCDM8
(Invitrogen, San Diego, CA). All other mutants were also cloned
into this vector. The deletion mutant 
202–304 was created by
cutting the cDNA of hTNF-R55 with EarI and
Eco57I, blunting both sticky ends with Klenow
polymerase and T4-exonuclease, respectively, and religating both
fragments. This process generated a receptor mutant with the following
cDNA sequence at the joint fragments: 5'-CTC CTC TTC ATT GCC
ATC CCC AAC-3'; the nucleotides filled in with Klenow polymeraseides
are shown in bold. The resulting protein lacks amino acids
203–303 of human TNF-R55, but still contains the FAN-binding domain
(14). Mutagenesis of leucine 351 to alanine (L351A) was performed
according to the instructions provided with the site-directed
mutagenesis kit (Clontech, Palo Alto, CA). The following mutator oligo
was used: 5'-GGT CGC TCG CCC CTA GGC GCC GCA C-3'. The mutant receptor
containing only the death domain (without the FAN-binding site) was
created as follows: a cDNA fragment containing the death domain of
human TNF-R55 preceded by a 15-aa linker (30) was ligated to the
extracellular and transmembrane part of human TNF-R55 and again
generated by cutting human TNF-R55 with EarI and treating
the fragment with Klenow polymerase. All mutants were verified by
sequencing.
Cell stimulation, immunocytochemistry, and microscopy
Experiments were performed at 37°C, each experimental
condition was tested on three different coverslips, and experiments
were repeated several times (2, 3, 4, 5). Where appropriate, cells were
preincubated with 0.3 µM of SB203580 for 120 min, 4 µM of cell
permeable ceramide for 30 min (New England Biolabs, Beverly,
MA), or 100 ng/ml human recombinant TNF (Genzyme, Cambridge,
MA) for 5 min, and subsequently stimulated with 100 ng/ml human
recombinant CSF-1 (Chiron Corporation, Emeryville, CA) for 10
min (J774.2 cells), 15 min (Bac1.2F5 cells and 4-4 cells), or 20 min
(P388D1 cells), or stimulated for 30 min with 1000 infectious
units/ml murine TNF (4-4 cells; murine TNF was produced in
Escherichia coli, purified to 
99% homogeneity in our
laboratory, and contained 4 ng of endotoxin/mg protein). For testing
the action of mutated human TNFRs, 4-4 cells (107
cells in 1 ml of RPMI 1640 containing 5% FCS) were transfected with a
total of 5 µg of cDNA by electroporation (300 V/950 µF/10 ms), and
seeded on coverslips. After 24 h, cells were stimulated for 30 min
with the htr-1 mAb (100 ng/ml; a gift of Dr. M. Brockhaus, Hoffman-La
Roche, Basel, Switzerland), which clusters and thus activates the
transfected human TNFRs but not the endogenous murine receptors (31, 32). To investigate pinocytosis, fluorescently labeled BSA (prepared
using Fluorlink; Amersham, Arlington Heights, IL) was added to
the culture medium in some experiments together with htr-1 Ab.
After stimulation, cells were fixed in 3.5% formaldehyde/PBS (v/v). For visualization of actin filaments, cells were permeabilized in 0.1% Triton X-100/PBS (v/v), blocked in 0.5% BSA (Sigma, St. Louis, MO)/PBS (w/v) for 45 min, and stained with 80 ng/ml tetramethylrhodamine B isothiocyanate (TRITC)-conjugated phalloidin (Sigma) in PBS for 1 h. For immunostaining, cells were blocked for 1 h in PBS containing 10% FCS (v/v), 5% milk powder (w/v), 0.5% BSA (w/v), and 0.1% Triton X-100 (v/v). Subsequently, cells were incubated in PBS containing 1% BSA (w/v) and 0.1% Triton X-100 (v/v) supplemented with a 1/2000 dilution of the htr-9 Ab (for the detection of transfected cells; the htr-9 Ab was also a kind gift of Dr. M. Brockhaus). Primary Ab binding was detected by incubation for 1 h with a 1/500 dilution of FITC-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS containing 1% BSA (w/v) and 0.1% Triton X-100 (v/v). Cell images were generated using confocal laser scanning microscopy as described previously (33, 34).
Microinjection
Protein expression, purification, and a determination of the
active protein concentration were performed as described previously
(26, 27). Microinjection were performed in RPMI 1640 without serum at
37°C. At least 30 cells were microinjected for every experimental
condition. Where relevant, 100 ng/ml TNF or 5 mM of C2-ceramide was
added to this medium. Cells were microinjected with 50 mM of Tris
buffer (pH 7.3 at 37°C) containing 100 mM NaCl, 5 mM
MgCl2, 150–700 µg of recombinant protein/ml, and 800
µg/ml rat IgG. After microinjection and, if appropriate, stimulation
with 100 ng/ml CSF-1, cells were fixed in 3.5% formaldehyde/PBS (v/v)
for 30 min. Subsequently, cells were blocked for 1 h in PBS
containing 10% FCS (v/v), 5% milk powder (w/v), 0.5% BSA (w/v), and
0.1% Triton X-100 (v/v) and stained with 80 ng/ml TRITC phalloidin and
a 1/500 dilution of FITC-conjugated rabbit anti-rat IgG (Jackson
Laboratories) in PBS containing 1% BSA (w/v) and 0.1% Triton X-100
(v/v). Microinjected cells were identified using the anti-mouse IgG
signal, and actin filaments were analyzed as described above.
Uptake of [3H]sucrose
Where appropriate, cells were preincubated for 16 h with 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA). Pinocytosis was assayed in RPMI 1640 containing 0.5 µCi of [3H]sucrose (Amersham). After the indicated incubation times, cells were washed five times with ice-cold RPMI 1640 and lysed in 1% SDS (w/v). Subsequently, cellular sucrose uptake was determined by scintillation counting. Each datapoint represents the average of at least three independent determinations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of TNF on actin organization in M were investigated
in four different mouse M cell lines to ensure that responses were
widely observed and not specific to only one cell line. To distinguish
between the effects mediated by TNFR-1 and TNFR-2, responses to both
human and mouse TNF were investigated, as human TNF can only stimulate
TNFR-1 in mouse cells (13). Unstimulated, serum-starved, Bac1.2F5 M
were rounded, and displayed relatively high levels of polymerized actin
within the cell interior (Fig. 1
A). Serum-starved J774.2
cells were more spread and elongated compared with Bac1.2F5 cells (Fig. 1D). Unstimulated P388D1 cells exhibited an elongated
phenotype with occasional microspikes; the majority of filamentous
actin (F-actin) was in the cell cortex (Fig. 1G). Upon
stimulation with either human or mouse TNF, all three M cell types
displayed a dramatic decrease in TRITC-labeled phalloidin staining. In
Bac1.2F5 cells, this effect was already observed after 5 min, was
maximal after 30 min, persisted for 2 h, and was somewhat more
pronounced at the cell periphery compared with the perinuclear region
(Fig. 1B). In J774.2 M cells, a decrease in F-actin was
first apparent at 15 min after TNF addition; a clear effect was
observed at 30 min (Fig. 1E). The decrease in F-actin in
P388D1 cells principally involved cortical actin structures (Fig. 1H) and followed a similar time course to that observed in
J774.2 cells. In 4-4 cells, TNF caused a response that was similar to
that observed in Bac1.2F5 cells (data not shown). We conclude that TNF
causes a reduction in F-actin in M and that TNFR-1 stimulation is
sufficient for this response.
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To gain insight into the signaling mechanisms mediating
TNF-dependent actin reorganization, we first investigated the effects
of D609. This compound inhibits a variety of death domain-dependent
responses to TNF (35). Treating P388D1 cells or J774.2 cells for 1
h with 20 µg/ml D609 did not detectably alter the actin organization
of these cells (data not shown). However, after subsequent stimulation
with TNF, the TNF-induced decrease in F-actin was not observed; an
accumulation of F-actin at the cell cortex was observed instead (Fig. 1
, C, F, and I). These data suggest an
involvement of the death domain-dependent phosphatidyl choline-specific
phospholipase C (35) in the loss of F-actin in response to TNF and also
indicate that TNF is able to induce actin reorganization at the cortex
when the overall decrease in F-actin is prevented.
To investigate further the signaling mechanisms underlying TNF-induced
actin reorganization, we transiently transfected mutants of human
TNFR-1 into mouse 4-4 M. Subsequent clustering of these transfected
receptors, using the Ab htr-1, initiated signaling through the
transfected receptors without activating endogenous murine TNFRs (31, 32). After fixation, the actin cytoskeleton of transfected cells was
compared with untransfected neighboring cells by double-staining the
cells with phalloidin and an Ab specific for human TNFR-1. Activation
of full-length human TNFR-1 in transfected cells led to a decrease in
phalloidin staining compared with untransfected cells (Fig. 2, A and B),
confirming that activation of TNFR-1 is sufficient to induce the
decrease in F-actin. Subsequently, we tested the effect of a TNFR-1
containing the L351A mutation (homologous to the lpr mutation in the
Fas Ag; 36), which renders the death domain inactive. Stimulation of
this receptor actually induced an increase rather than a decrease in
F-actin (Fig. 1, I and J); however, in contrast
to the effect of D609, this increase was not limited to the cell cortex
(possibly because the higher expression of transfected TNFR compared
with the endogenous receptor). Surprisingly, activation of a TNFR
lacking the membrane proximal part of the receptor but containing a
functional death domain and a FAN-binding domain (deletion of amino
acids 202–304) also resulted in increased levels of F-actin. A
receptor mutant lacking both the membrane proximal part and Fan-binding
site but containing a functional death domain (see Materials and
Methods) did not induce any detectable actin reorganization
(Fig. 2, E and F). Therefore, loss of polymerized
actin in response to TNFR-1 activation requires both the membrane
proximal part of the receptor and a functional death domain, whereas
the FAN-binding site appears to mediate a TNF-induced increase in
F-actin.
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Macropinocytosis can be stimulated in M by a number of stimuli,
including phorbol esters, and involves actin reorganization (37). TNF
induced the accumulation of vesicles in M (Fig. 1B), an
effect that was sensitive to D609 (Fig. 1C). To test whether
these vesicles were a result of a TNF-induced increase in
macropinocytosis, we transfected 4-4 M with human TNFR-1 and
compared the uptake of fluorescently labeled BSA by transfected cells
with that of untransfected cells. After stimulation with the Ab htr-1,
human TNFR-1-expressing cells showed a considerably higher uptake of
fluorescent BSA compared with neighboring cells (Fig. 2, B
and C). Subsequently, we performed experiments to establish
the domains of TNFR-1 necessary for this effect of TNFR-1. A receptor
mutant in which both the membrane proximal part and the FAN-binding
site were deleted was still able to mediate an increased uptake of
fluorescent BSA (Fig. 2, G and H), whereas TNFR-1
containing the L351N mutation (and thus having an inactive death
domain) was unable to induce an increased BSA uptake (Fig. 2, K and L). We conclude that increased
macropinocytosis in response to TNF is solely mediated by the death
domain of the receptor.
Phorbol esters regulate TNF-induced macropinocytosis
To quantify the extent of TNF-induced macropinocytosis, the
pinocytotic activity in J774.2 M was assayed directly by measuring
[3H]sucrose uptake. Incubating cells with 100 ng/ml mouse
TNF caused an increase in the rate of fluid uptake, and this effect
persisted for 1 h (Fig. 3A).
Subsequent dose-response experiments established that an effect of TNF
on fluid uptake was already observed at TNF concentrations in excess of
10 ng/ml, whereas the response was maximal at 50 ng/ml (Fig. 3B). The inhibition of TNF-induced macropinocytosis by D609
suggested that stimulation of phosphatidylcholine-specific
phospholipase C could be involved in this response (35). As
phospholipase C activation leads to the production of diacylglycerol
and to the activation of several protein kinase C (PKC) enzymes (38)
and as PKCs are known to regulate macropinocytosis (37), the effect of
the PKC activator TPA on macropinocytosis was investigated. The
addition of 100 ng/ml TPA potently stimulated fluid uptake in J774.2
M (Fig. 4A). To investigate
whether PKC is involved in the TNF response, cells were treated with
TPA for 16 h to down-regulate phorbol ester-responsive PKC
isoforms. A subsequent application of TPA was no longer able to
stimulate macropinocytosis (Fig. 4A). Interestingly,
TNF-induced macropinocytosis was also inhibited after TPA pretreatment
(Fig. 4B). Taken together, these results strongly suggest
that PKC or another phorbol ester-binding protein mediates
TNF-stimulated macropinocytosis.
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TNF can inhibit phagocyte chemotaxis (39), and we have
specifically shown that TNF inhibits the chemotaxis of Bac1.2F5 cells
toward CSF-1 but has no effect on CSF-1-induced chemokinesis (11). To
investigate the mechanisms underlying the ability of TNF to inhibit
chemotaxis, we decided to characterize the effects of TNF on
CSF-1-induced actin remodeling. CSF-1 induces rapid actin
reorganization in Bac1.2F5 cells: it stimulates the formation of
filopodia, lamellipodia, and membrane ruffles (28). A similar response
to CSF-1 is observed in J774.2, P388D1, and 4.4 M (Fig. 5, A, E, and
I).
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, CSF-1 was still able to induce substantial
reorganization of the actin cytoskeleton; however, filopodium formation
was almost completely inhibited. In contrast, CSF-1-induced
lamellipodium production and ruffling were enhanced by TNF pretreatment
in Bac1.2F5 (Fig. 5C), J774.2 (Fig. 5G), and
P388D1 cells (Fig. 5K). Similar effects were observed when
TNF and CSF-1 were added at the same time (data not shown). We conclude
that TNF specifically inhibits CSF-1-induced filopodium formation,
whereas lamellipodium formation and ruffling are not sensitive to TNF. TNF inhibits filopodium formation downstream of Cdc42
As Cdc42 mediates CSF-1-induced filopodium extension in M (28),
the question arises as to whether TNF inhibits Cdc42 activation or
whether it interferes with the signaling pathway leading from activated
Cdc42 to the cytoskeleton. To distinguish between these two
possibilities, J774.2 M were microinjected with Cdc42 V12 (a
constitutively active mutant of Cdc42) in the presence or absence of
TNF. As expected, Cdc42 V12 induced a large increase in filopodium
formation (Fig. 6A),
but this response was completely inhibited by TNF (Fig. 6B).
In contrast, TNF did not influence the actin reorganization induced by
a microinjection of Rac V12 and Rho V14. Therefore, TNF specifically
interferes with the signal transduction pathway leading from activated
Cdc42 to filopodium production.
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TNF is known to activate members of the MAPK family of proteins
(40). Therefore, we investigated the roles of p42/44 MAPK and p38 MAPK
in TNF-induced actin reorganization using the MAPK/extracellular
signal-regulated kinase inhibitor PD098059 (41) and SB203580, a
p38 MAPK inhibitor (42). PD098059 or SB203580 did not alter the
TNF-induced decrease in F-actin or macropinocytosis (Table I).
However, SB203580 (Fig. 5) but not PD098059 (data not shown) was able
to completely abrogate the inhibitory effect of TNF on CSF-1-induced
filopodium production in P388D1 cells and partial restored the response
in Bac1.2F5 and J774.2 M. Furthermore, treatment of cells with 4
µM of C2-ceramide, which potently activates p38 MAPK, reduced CSF-1-
and Cdc42-dependent filopodium production (Fig. 6C). We
conclude that the inhibitory effect of TNF on filopodium production is
mediated at least in part by p38 MAPK.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, but its effects on
the M actin cytoskeleton have not been studied in detail. We report
here that TNF induces a decrease in polymerized actin and an increase
in macropinocytosis and inhibits Cdc42-mediated filopodium extension.
In addition, when the TNF-induced decrease in F-actin is inhibited
using D609 or receptor mutants, a distinct type of actin reorganization
involving an increase in F-actin is observed in response to TNF. For
all of these effects of TNF, activation of TNFR-1 was sufficient, as
specific activation of transfected TNFR-1 by the htr-1 Ab induced all
of the responses observed with TNF. Similarly, these responses to TNF
could be elicited by adding human TNF to mouse M, which will result
in the activation of only mouse TNFR-1 and not mouse TNFR-2 (13).
Consequently, TNFR-2 does not appear to be required for TNF-induced
cytoskeletal reorganization in mouse M.
Transfection of specific TNFR-1 mutants allowed us to pinpoint the
elements in the receptor mediating the different effects of TNF on the
actin cytoskeleton (Fig. 7). We observed that for the TNF-induced
decrease in F-actin, both the membrane proximal region of TNFR-1 and
the death domain were essential. The requirement for these two domains
is unusual and has not been observed for most other TNF-induced
responses, for which either the death domain or the FAN-binding site is
sufficient; interestingly, TNF-induced induction of nitric oxide
synthase also requires these two domains of TNFR-1 (21). In addition,
L929 cells transfected with a human TNFR-1 lacking the membrane
proximal region display markedly delayed cell death in response to the
htr-1 Ab as compared with the full-length receptor, although NF-B
activation was not affected (43). Apparently, the membrane proximal
part of the receptor exerts important cooperative functions with the
death domain of the receptor, and may be more important in TNF
signaling than previously thought.
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actin cytoskeleton, to determine their role in
TNF-induced cytoskeletal remodeling, and to assess possible interplay
between the effects of these proteins.
When the TNF-induced decrease in F-actin was inhibited by the compound
D609 or by activating TNFR mutants lacking either the membrane proximal
region or the death domain, a TNF-induced increase in F-actin was
observed. This finding suggests that the signal inducing the decrease
in F-actin is sufficiently strong enough to override other signals from
TNFR-1 to the actin cytoskeleton, but that when this signal is reduced,
less dominant signals can induce a distinct type of actin
reorganization. One possibility is that TNF activates a protein that
enhances actin filament turnover, such as ADF/cofilin
(47), which then rapidly depolymerizes any
actin-containing structures produced in response to other TNF-activated
signals. According to this model, these other structures would be more
stable when the rate of actin depolymerization is decreased. The
TNF-induced increase in F-actin was mechanistically distinct from the
decrease in F-actin, as it was dependent upon the FAN-binding site of
the receptor. The signaling pathways downstream of FAN are still
unclear but include activation of neutral sphingomyelinase activity,
generating ceramide (14). However, an application of cell-permeable
ceramides did not induce an increase in F-actin (Fig. 6), indicating
that ceramide production is not sufficient for this response. The
signaling mechanisms by which the FAN-binding site mediates actin
reorganization in M may well involve Rho family proteins; therefore,
we plan to investigate this possibility.
We observed that TNF was a potent stimulator of macropinocytosis. For
this effect, a functional death domain was sufficient, and D609 also
inhibited the macropinocytosis induced by TNF. The molecular target for
D609 is apparently a TNF-activated phosphatidylcholine-specific
phospholipase C, which leads to PKC activation through diacylglycerol
production (35). Phorbol esters, which mimic the action of
diacylglycerol and activate PKC, potently stimulate macropinocytosis in
M (37); down-regulation of PKC by prolonged treatment with phorbol
esters impaired TNF-induced pinocytosis. These results suggest that a
phorbol ester-sensitive isoform of PKC is required for TNF-induced
macropinocytosis. However, D609 also inhibited phorbol ester-dependent
pinocytosis in M (M.P.P., unpublished observations), suggesting that
this compound acts downstream rather than upstream of diacylglycerol
release to inhibit pinocytosis and consequently has multiple cellular
targets. It is also possible that another phorbol ester-binding protein
apart from PKC is involved in the macropinocytic response (for example,
a member of the chimerin protein family that acts as a GTPase
activating protein for Rac and Cdc42) (48). Indeed, expression
of activated Rac has been shown to stimulate macropinocytosis in
fibroblasts (27).
Macropinocytosis can act as a nonspecific mechanism for Ag
capture, leading to Ag presentation and T cell activation (37). TNF is
a potent inducer of Ag presentation in M (6), and TNF-stimulated
macropinocytosis may therefore be important for this response; current
experiments address this possibility. Alternatively, TNF-induced
macropinocytosis may serve to translocate activated receptors into
intracellular compartments, as it has been shown that a number of TNF
effects require TNFR internalization and the subsequent acidification
of the endosomal compartment (35), although this scheme makes the
effects of membrane-bound TNF, which is not internalized, difficult
to explain. Strikingly, the TNF responses that require endosomal
acidification are identical with those that are inhibited by D609.
Therefore, we speculate that D609 may interfere with TNF
signaling, at least in part, by inhibiting TNF-induced
macropinocytosis and thus preventing TNFR translocation to
endosomes.
TNF inhibits chemotaxis of phagocytes without affecting chemokinesis
(6, 7, 8, 9, 10, 11, 39). To investigate the cytoskeletal basis of this inhibition,
we tested the effects of TNF on CSF-1-induced actin remodeling. We
recently demonstrated that CSF-1 induces Cdc42-mediated filopodium
formation and Rac-mediated lamellipodium formation in M (28). In
this study, we observed that TNF inhibited Cdc42- and CSF-1-induced
filopodium production but did not impair Rac- or CSF-induced
lamellipodium extension. Filopodia have been suggested to play a
pivotal role in chemotaxis as sensors for the chemotactic gradient;
thus, the TNF-induced inhibition of Cdc42-mediated filopodium extension
may explain the inhibitory effect of TNF on M chemotaxis. The signal
leading to an overall decrease in F-actin in response to TNF may
contribute to the inability of cells to produce filopodia, although it
clearly does not prevent membrane ruffling.
The inhibitory effect of TNF on filopodium extension mapped to the death domain of TNFR-1. Activation of p38 MAPK by TNF is similarly dependent upon this domain (40), and in the presence of the p38 MAPK inhibitor SB203580, TNF no longer blocked filopodium production, suggesting that p38 MAPK mediates the inhibition of filopodium formation. In agreement with this, we observed that cell-permeable ceramides, which stimulate p38 MAPK, impaired Cdc42-induced filopodium formation. Interestingly, both actinomycin D and UV light strongly activate p38 MAPK in most cells, and have been reported to inhibit leukocyte chemotaxis (49, 50). This effect may also be due to p38 MAPK-mediated inhibition of filopodium formation. As the death domain of TNFR-1 mediated both macropinocytosis and an inhibition of filopodium formation, it is possible that macropinocytosis is causally associated with the lack of filopodia. However, SB203580 did not block macropinocytosis, and therefore these two responses mediated by the death domain are separable. Presumably, another target(s) of the death domain is involved in mediating the macropinocytotic response to TNF.
TNF is one of the most pleiotropic cytokines known, and its
effects on cell migration and locomotion vary considerably between
different cell types; for instance, TNF inhibits M chemotaxis but
acts as a chemoattractant for fibroblasts (51). Our results show that
distinct signals generated by different regions of TNFR-1 have specific
effects on actin organization in M (Fig. 6). The membrane proximal
region of the receptor together with the death domain is required for
the decrease in F-actin, whereas the FAN-binding site is required for
distinct changes in actin organization involving a localized increase
in F-actin. The death domain alone is sufficient to mediate
macropinocytosis and an inhibition of filopodium formation. Hence,
different TNF-activated signal transduction pathways can exert
contrasting effects on the M actin cytoskeleton, with the final
response presumably being dependent upon the relative abundance of
secondary mediators and the strengths of these distinct signals.
Consequently, differential regulation of these signaling pathways may
explain the specific actions of TNF on cell locomotion in different
cell types.
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Footnotes |
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2 Abbreviations used in this paper: M, macrophage(s); TNFR, TNF receptor; TPA, 12-O-tetradecanoylphorbol-13-acetate; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; PAK, p21-activated kinase; TRITC, tetramethylrhodamine B isothiocyanate.
Received for publication May 28, 1998. Accepted for publication October 5, 1998.
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and dotted line) or with 100 ng/ml mouse
TNF (• and solid line) for the times indicated, and sucrose uptake
was determined. B, Dose-dependent stimulation with mouse
TNF. Cells were stimulated with the TNF concentrations indicated;
[3H]sucrose uptake was determined after 60
min.
and solid line) or treated with TPA
for 16 h and then restimulated with TPA (
and dashed line).
[3H]sucrose uptake was determined. B,
[3H]sucrose uptake induced by 100 ng/ml mouse TNF in
control cells (• and solid line) and in cells treated for 16 h
with 100 ng/ml TPA (
