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INTRODUCTION |
Tumor necrosis factor
(TNF),1 originally defined by
its antitumoral activity, is now recognized as a pleiotropic cytokine
exerting a wide variety of immunoregulatory activities (for review, see Refs. 1-3). TNF action is mediated by two types of cell surface receptors of 55 kDa (TR55) and 75 kDa (TR75) molecular masses, respectively. Both receptors mediate distinct TNF responses (4-6). The
majority of activities of soluble TNF appears to be mediated by TR55
(7-11). Like other cytokine receptors, the cytoplasmic domain of both
TNF receptors lacks intrinsic enzymatic activities. The activation of
intracellular signaling enzyme systems is initiated by a selective
interplay between the cytoplasmic domain of the TNF receptors and a
number of recently identified TNF receptor-associated proteins (see
Ref.12 for review). The adapter protein TRADD associates with the
so-called death domain of TR55, recruiting FADD, RIP, and TRAF-2
(13-16). FADD mediates the activation of a protease termed FLICE/MACH
(17, 18), representing one of the first steps in induction of the
apoptotic pathway. RIP mediates activation of the transcription factor
NF-
B associated with anti-apoptotic regulatory functions (19).
TRAF-2 links TR55 to activation of the N-terminal c-Jun kinase (JNK)
cascade (20). The TR55-associated protein FAN (21) binds to a domain
designated neutral sphingomyelinase activation domain (NSD) that is
N-terminally adjacent to the death domain (22). FAN mediates
TNF-induced activation of neutral sphingomyelinase (N-SMase) (21).
Results from numerous studies have revealed that TNF signaling further
involves activation of downstream enzyme systems at multiple
subcellular compartments such as the plasmamembrane, endosomes,
mitochondria, the cytosol, and the nucleus (for review, see Refs. 11,
23, and 24). Membrane-associated enzyme systems transmitting TR55
signals include plasmamembrane-bound phospholipases such as
phosphatidylcholine-specific phospholipase C (25), which generates the
lipid second messenger molecule 1,2-diacylglycerol and a N-SMase (9),
producing ceramide by sphingomyelin hydrolysis. Ceramide generated at
the plasmamembrane triggers activation of a 97-kDa ceramide-activated
protein kinase (26), recently suggested to be identical with the
"kinase suppressor of ras" (27). Ceramide-activated protein kinase belongs to a family of proline-directed protein kinases
(PDPK) (9), including members of the mitogen-activated protein kinases
(28). TNF signaling further involves intracellular membrane
compartments like caveolae and endosomes harboring an acid SMase
(A-SMase) (9, 29, 30). In mitochondria, TNF induces reactive oxygen
species that are generated at the level of the oxidative
phosphorylation complex III (31). The induction of the mitochondrial
permeability transition has been linked to the TNF cytotoxic pathway
(32). In addition, cytosolic protein kinase cascades including PKC and
JNK have been identified to transmit TNF signals (33-35).
The question of how TNF signals are targeted to the different
intracellular compartments is of fundamental biological significance. Intriguingly, rapidly diffusible ions like Ca2+ seem not to
be involved in TNF signaling. Rather, the two lipid second messenger
molecules 1,2-diacylglycerol and ceramide are likely to reside within
membranes because of their hydrophobic nature. Thus the mechanisms of
intracellular TNF signal trafficking remain obscure.
In the present study we investigated the effects of inhibition of TNF
receptor endocytosis on TNF signal transduction. Because structural
motifs within the TNF receptor required for endocytosis are currently
unknown, genetic approaches to block TNF receptor internalization are
not available at present. We here employed the primary amine
monodansylcadaverine (MDC) or K+ depletion to inhibit TNF
receptor internalization. MDC is an inhibitor of transglutaminase, a
membrane-bound enzyme that actively participates in internalization of
various receptor systems (36-44).
We show here that MDC as well as K+-depletion block TNF
endocytosis, which is related to inhibition of TR55 death domain
signaling like TNF-dependent activation of endosomal
A-SMase, JNK, and TNF-mediated apoptosis. In contrast,
TNF-dependent stimulation of
plasmamembrane-associated N-SMase and PDP kinase are not affected by
MDC or K+ depletion.
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MATERIALS AND METHODS |
Cell Culture and Reagents--
U937, Jurkat, and HeLa cells were
obtained from the American Type Culture Collection, Manassas, VA, and
maintained in a mixture of Click's/RPMI 1640 (50%, 50% v/v)
supplemented with 5% fetal calf serum, 10 mM glutamine,
and 50 µg/ml each streptomycin and penicillin in a humidified
incubator containing 5% CO2. The human embryonic kidney
cell line HEK 293 was kindly provided by Dr. M. Schmidt, Essen, Germany
and maintained in high glucose Dulbecco's modified Eagle's medium
(ICN), 10% fetal calf serum, 10 mM glutamine, and 50 µg/ml each streptomycin and penicillin. The cDNA for gluthathione S-transferase (GST)-Jun (1-166) was provided by Dr. P. Angel (DKFZ, Heidelberg, Germany). Highly purified recombinant human
TNF (3 × 107 units/ml) was provided by Dr. G. Adolf,
Bender Research Institute Vienna, Austria. Monodansylcadaverine was
obtained from Sigma, and C6-ceramide
(N-hexanoylsphingosine) was purchased from Biomol Feinchemikalien GmbH, Hamburg, Germany. Anti-CD95 (Fas/APO-1) was
obtained from Calbiochem-Novabiochem GmbH, Bad Soden, Germany. A pRK5
expression plasmid encoding TRADD was kindly provided by Dr. D. V. Goeddel, Tularik Inc., S. San Francisco, CA. The monoclonal anti-TR55
antibody htr9 was a gift from Dr. W. Lesslauer and Dr. H. Loetscher,
Hoffmann-La Roche, Basel, Switzerland.
Measurements of TNF Receptor-mediated Internalization--
One
million U937 cells were incubated for 1 h at 0 °C with 1 ng of
125I-labeled human recombinant TNF (NEN Life Science
Products, specific activity 2160 kBq/µg) to saturate cell surface TNF
receptors. After washing the cells three times in cold PBS, the
temperature was shifted to 37 °C for 1 h to allow receptor
internalization. To determine the amount of internalized
125I-TNF receptor complexes, noninternalized ligand was
removed by passing cells through a pH 3 gradient at 500 × g consisting of (a) 0.5 ml of culture medium
supplemented with 20% Ficoll and (b) 3 ml of 100 mM NaCl, 50 mM glycine/HCl, pH 3, supplemented with 10% Ficoll, and 0.5 ml of culture medium containing 5% Ficoll. To determine the total amount of cell-associated 125I-TNF,
a second aliquot of cells was passed through a gradient in which the
second layer (b) was replaced by PBS, pH 7.3, containing 10% Ficoll. Radioactivity in the cell pellets was determined by liquid
scintillation counting. To measure nonspecific binding, a parallel
experiment was performed in which a 200-fold excess of unlabeled TNF
was added to the cells. Specific binding was calculated by subtracting
nonspecific from total binding, and the amount of 125I-TNF
internalized was calculated as the percent of specific binding determined at pH 7.3.
Fluorescence Microscopy of TNF-Biotin/Avidin-FITC-labeled
Cells--
HeLa cells were grown on glass coverslips in Click's RPMI
medium and either left untreated or were incubated with 100 µM MDC for 1 h at 37 °C. Cells were then shifted
to 4 °C, and 100 ng/ml biotinylated TNF (human recombinant TNF-
biotin conjugate, Fluorokine, R & D Systems, Wiesbaden) was added.
After 2 h, cells were washed with PBS to remove unbound TNF, one
set of cells was fixed with ethanol as a control of surface staining,
and the other cells were shifted to 37 °C, and biotin-TNF bound to
the TNF receptor was allowed to internalize for 10, 30, and 60 min.
Cells were fixed with cold methanol, avidin-FITC (1:40 final dilution)
was added for 1 h at 37 °C, and fluorescence was documented
using a fluorescence microscope (Zeiss). The biological activity of biotinylated TNF was identical to nonmodified TNF as estimated in MTT
cytotoxicity assays, indicating that biotinylation of recombinant TNF
did not alter its receptor binding properties
Inhibition of TNF Receptor Internalization--
Low temperature
effects were assayed by incubation of cells at 14 °C for 1 h.
Depletion of intracellular K+ was performed as described
(45). Briefly, cells were washed twice in buffer A (100 mM
NaCl, 50 mM Hepes, pH 7.4) and subjected for 5 min to
hypotonic medium (medium/water 1:1) followed by a 10 min incubation in
buffer A. All treatments were performed at 37 °C. The cells were
then resuspended in buffer B (100 mM NaCl, 1 mM
CaCl2, 50 mM Hepes, pH 7.4) and kept at
37 °C for an additional 30 min. The effect of MDC was estimated by
treating cells with 100 µM MDC for 1 h in serum-free
medium supplemented with 2% bovine serum albumin.
Assays for Neutral and Acid Sphingomyelinase--
Micellar SMase
assays using exogenous radiolabeled sphingomyelin as substrate was
performed according to a method previously described (9, 46). Briefly,
U937 cells (3 × 106/ml) in triplicates were
homogenized, and 50 µg of protein from the cellular lysates were
examined for SMase activity using a micellar assay system with
[N-methyl-14C]sphingomyelin (0.2 µCi/ml,
specific activity 56.6 mCi/mmol, Amersham Pharmacia Biotech) as a
substrate. [14C]Phosphorylcholine, produced from
[14C]sphingomyelin, was extracted from the aqueous phase,
identified by TLC, and routinely determined by liquid scintillation counting.
JNK Assay--
Cell extracts were prepared by homogenization in
lysis buffer (consisting of 25 mM HEPES, pH 7.55, 100 mM NaCl, 1, 5 mM MgCl2, 0.5 mM EGTA, 0.25 mM EDTA, 10 mM NaF,
0.1% Nonidet P-40, 20 mM
-glycerophosphate, 1 mM vanadate, 1 mM phenylmethylsulfonyl
fluoride, and 10 µg/µl each of leupeptin, pepstatin, and aprotinin)
followed by centrifugation for 30 min at 20,000 × g.
For assessment of JNK activity, 100 µg of supernatant was
immunoprecipitated with 2 µg of affinity-purified polyclonal
anti-JNK-1 antibody (C-17) obtained from Santa Cruz Biotechnology,
Inc., Santa Cruz, CA. Immunecomplexes were recovered by protein
G-Sepharose (Amersham Pharmacia Biotech), washed extensively, and
assayed for in vitro kinase activity using 5 µg of GST-Jun
1-166 as a substrate. Phosphorylation was performed for 20 min at
30 °C in 20-µl assays consisting of 50 mM HEPES, pH
7.55, 100 mM KCl, 25 mM
-glycerophosphate,
10 mM MgCl2, 1 mM
MnCl2, 1 mM Na3VO4, 0.5 mM EGTA, 20 µM ATP, and 0.5 µCi of
[
-33P]ATP (specific activity > 2000 Ci/mmol).
Phosphoproteins were analyzed on 12% SDS-polyacrylamide gel
electrophoresis followed by autoradiography and two-dimensional laser
scanning (Molecular Dynamics Personal Densitometer).
PDPK Assay--
PDP kinase was assayed as described (9).
Briefly, cell extracts were prepared by homogenization in cold
extraction buffer consisting of 20 mM HEPES, pH 7.4, 10 mM MgCl2, 2 mM EDTA, 10 mM NaF, 10 mM
-glycerophosphate, 0.1 mM vanadate, 0.1 mM molybdate, 1 mM
phenylmethylsulfonyl fluoride, and 10 µg/µl each of leupeptin, pepstatin, and aprotinin), followed by centrifugation for 30 min at
20,000 × g. For assessment of PDPK, 10 µg of lysate
in 10 µl was mixed with 10 µl of reaction buffer consisting of 60 mM Hepes, pH 7.4, 30 mM MgCl2, 1 µCi of [
-33P]ATP (1000 Ci/mmol), and 10 µl (6 mg/ml) of peptide
Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 as substrate.
The reaction mixture was incubated for 20 min at 30 °C and stopped
by the addition of 4 µl of 88% formic acid. Samples were centrifuged
at 20,000 × g for 30 min at 4 °C. 20-µl aliquots
of the supernatants were spotted onto 2.5-cm diameter circles of
phosphocellulose paper and washed in four changes of 1%
H3PO4, and radioactivity was estimated by
scintillation counting.
Transient Expression Experiments and in Vivo Interaction
Assay--
For transient expression of FLAG-tagged FAN and TRADD,
1.5 × 106 HEK 293 cells were seeded on 100-mm dishes
and transfected with pRK FLAG-TRADD or pFLAG.CMV2-FAN the following day
by the calcium phosphate precipitation method. After 18 h of
incubation, cellular extracts were prepared and immunoprecipitated
using the anti-TR55 antibody htr-9 as described (21).
Immunoprecipitated proteins were separated by 12.5% SDS-polyacrylamide
gel electrophoresis and blotted on nitrocellulose membranes. Western
blots were performed using anti-FLAG antibody M5 (Kodak International
Biotechnologies), a peroxidase-coupled rabbit-anti-mouse antiserum
(Dianova), and the ECL immunodetection system (Amersham Pharmacia Biotech).
Cytotoxicity Assay--
Cell viability was assessed by using the
MTT conversion assay, performed in quadruplicates in microtiter plates
at a cell density of 2 × 104 cells/well. U937 cells
were either left untreated at 37 °C or subjected to
K+ depletion, MDC treatment, or were kept at 14 °C to
block TNF receptor internalization. TNF at various concentrations was
added for 24 h. 20 µl of MTT (Sigma, 2.5 mg/ml in PBS) was
added, and incubation was continued for an additional 2 h to allow
metabolization of MTT to
3-[4,5-dimethyldiazol-2-yl]-2,5-diphenylformazan, which was
solubilized with isopropanol-HCL (24:1) and colorimetrically determined at 570 nm in a microplate reader (MWG-Biotech).
Evaluation of Apoptosis--
Cytochemical staining. To estimate
nuclear morphology, the fluorescent DNA staining dyes acridine orange
and ethidium bromide were utilized as described (47). Approximately
1 × 106 cells in 25 µl were stained by adding 1 µl of a mixture containing 100 µg/ml acridine orange and 100 µg/ml ethidium bromide (Sigma) in PBS. Uptake of the dye was examined
by fluorescence microscopy. To estimate the apoptotic index, a minimum
of 300 cells was examined by at least two independent investigators and
quantified by recording the number of cells in the following groups: 1)
viable cells with normal nuclei showing bright green chromatin with
organized structure; 2) early and late apoptotic cells showing bright
green or orange chromatin, respectively, that is highly condensed or
fragmented, and 3) necrotic cells with bright orange nuclei with
organized chromatin structure.
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RESULTS |
Inhibition of TNF Receptor Internalization--
TR55 receptors are
rapidly internalized upon binding of the ligand via the classical
receptor-mediated endocytic pathway, involving clathrin-coated pit
formation (48). Notably, only TR55 is internalized upon TNF binding,
whereas TR75 is shed from the cell surface (49, 50). To investigate the
functional role of TR55 endocytosis for intracellular signal
transduction and processing of the apoptotic signal, TNF receptor
internalization was blocked by the transglutaminase inhibitor MDC or by
disrupting clathrin-coated pit formation by depleting cells from
intracellular K+.
In a first approach, the inhibitory effects of MDC on TNF receptor
endocytosis in U937 cells were analyzed by measuring endocytosis of
125I-labeled TNF and compared with effects of
K+ depletion or "freezing" membranes at 14 °C. As
shown in Fig. 1A, pretreatment
of U937 cells with 100 µM MDC, K+ depletion,
or low temperature resulted in an inhibition of TNF-induced receptor
internalization by 58.8, 64.4, or 74%, respectively. These values
correspond well to those previously obtained with endothelial cells
(41) and human skin fibroblasts (51).

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Fig. 1.
A, inhibition of TNF receptor
internalization by K+ depletion, MDC treatment, or low
temperature. U937 cells were either left untreated or incubated with
100 µM MDC for 1 h, depleted (depl.) of
intracellular K+, or kept at 14 °C. Cells were then
incubated for 2 h with 125I-TNF (10 ng/ml) at 4 °C.
Afterward, cells were shifted to 37 °C, and TNF receptor
internalization was determined as described under "Materials and
Methods." The mean values from three experiments performed in
triplicates (± SD) are shown. B, MDC
dose-dependent inhibition of TNF receptor internalization.
U937 cells were preincubated with the indicated concentrations of MDC,
and TNF receptor internalization was determined as in A
(n = 3, ±S.D.).
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The inhibitory effect of MDC on TNF receptor internalization was
dose-dependent (Fig. 1B), with half-maximal
inhibition at 30 µM MDC. This value is consistent with
the half-maximal MDC concentration required for inhibition of
transglutaminase, an enzyme crucially involved in coated pit formation,
the initial step in receptor endocytosis (37).
To demonstrate the effect of MDC on intracellular TNF receptor
distribution by an independent method, we subsequently employed biotinylated TNF/avidin-FITC to stain TNF receptors on HeLa cells and
to follow the internalization of the ligand upon incubation at
37 °C. Analysis of TNF binding on HeLa cells by indirect
fluorescence microscopy revealed a uniform surface staining of
TNF-biotin/avidin FITC complexes at 4 °C (Fig.
2a). Shifting the incubation
temperature to 37 °C for 10 min resulted in a pattern of condensed
fluorescence, typical of multimerized, clustered receptors most likely
located in early endosomal vesicles (Fig. 2b). After 30 min
at 37 °C, the vesicular staining appeared concentrated in larger
intracellular compartments (Fig. 2c), indicating
accumulation of TNF in late endolysosomal vesicles. After 60 min, a
perinuclear staining was observed colocalizing with lysosomal
compartments (Fig. 2d). These kinetics of intracellular
staining indicate uptake of receptor-bound TNF and transport of the
ligand to lysosomes within 1 h, an observation that is in line
with electron microscopic evaluations (48). In contrast, preincubation
of the cells with MDC completely blocked the intracellular
translocation of TNF in endolysosomal or perinuclear lysosomal
compartments as revealed by the uniform surface staining with
TNF-biotin/avidin FITC at 10, 30, and 60 min. (Fig. 2,
e-g). These observations suggested that TNF receptor
internalization and subsequent intracellular trafficking was prevented
by MDC.

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Fig. 2.
MDC blocks uptake of biotinylated TNF in HeLa
cells. HeLa cells were grown on glass coverslips and either left
untreated (a-d) or were incubated with 100 µM
MDC (e-g) for 1 h at 37 °C. Cells were then shifted
to 4 °C, and 100 ng/ml biotin-TNF was added. After 2 h, the
cells were washed with PBS, and 1 aliquot was fixed with cold methanol
as the control of surface staining (a). The remaining cells
were shifted to 37 °C, and biotin-TNF bound to the TNF receptor was
allowed to internalize for 10 min (b and e), 30 min (c and f), and 60 min (d and
g). Cells were fixed and stained with avidin-FITC, and
fluorescence was analyzed using fluorescence microscopy (magnification
100×).
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Activation of A-SMase but Not N-SMase Is Blocked by MDC and after
K+ Depletion--
Activation of acid and neutral SMases
are early TNF-responsive events (9) regulated by the death
domain-associated protein TRADD (52) and the neutral sphingomyelinase
activation domain (NSD)-associated protein FAN (21), respectively. As
shown in Fig. 3A, TNF
stimulation of A-SMase was completely blocked after pretreatment of
cells with MDC. The effect of MDC on A-SMase activation did not result
from direct inhibition of the enzyme, because this agent did not
inhibit A-SMase when added directly to A-SMase assays (data not shown).
The stimulation of A-SMase was also inhibited when cells were deprived
of K+ to block TNF receptor endocytosis. Thus, MDC-mediated
inhibition of A-SMase is likely secondary to the blockade of TNF
receptor internalization.

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Fig. 3.
A, MDC and K+ depletion
prevents TNF induction of acid SMase. U937 cells were left untreated
( ), pretreated with MDC ( ), or were depleted of intracellular
K+ ( ). Aliquots were incubated with 100 ng/ml TNF for
the indicated periods of time. Nuclei-free lysates were prepared and
assayed for A-SMase activity using
[14C]sphingomyelin as substrate. Basal A-SMase
activity corresponded to production of 1.05 nmol of
phosphorylcholine/mg of protein/h. TNF-stimulated SMase
activities are expressed as percent of control. Shown are the
mean values (±S.D.) of 3-5 experiments, each performed in
triplicates. B, MDC and K+ depletion does not
block TNF activation of neutral SMase. U937 cells were treated as in
A and analyzed for activation of N-SMase as described under
"Materials and Methods." Basal N-SMase activity corresponds to
production of 0.30 nmol of phosphorylcholine/mg of protein/h produced.
Shown are the mean values (±S.D.) of three experiments, each performed
in triplicates.
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In contrast, preincubation of U937 cells with MDC did not prevent
TNF-induced activation of N-SMase (Fig. 3B). Blocking TNF receptor internalization by K+ depletion also did not
prevent N-SMase activation. These data indicate that activation of the
plasmamembrane-associated N-SMase occurs independent of TNF receptor internalization.
Differential Requirements of Cytosolic Protein Kinases for TNF
Receptor Internalization--
TNF induces activation of PDPK including
a ceramide-activated protein kinase (53) and mitogen-stimulated protein
kinases (54). The stimulation of these protein kinases is possibly
secondary to ceramide generated by N-SMase (9, 54). As shown in Fig. 4, TNF induction of PDP kinase activity
was not inhibited by MDC. In contrast, pretreatment with MDC resulted
in a marked inhibition of TNF-induced activation of JNK (Fig.
5, b and c).
Notably, treatment of cells with exogenous C6-ceramide
overcame the MDC-imposed blockade of JNK activation (Fig. 5,
e and f). Similar effects of MDC on PDP kinase
and JNK were also observed with K+-depleted cells
(Figs. 4 and 5, c and f). Taken together, these findings suggest that the cytosolic protein kinases JNK and PDPK are
activated by separate pathways that are either dependent or independent
of TNF receptor internalization.

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Fig. 4.
TNF-dependent induction of PDP
kinase is not prevented by MDC or K+ depletion. U937
cells were left untreated ( ), pretreated with MDC ( ), or
K+-depleted ( ) before stimulation with 100 ng/ml TNF for
the indicated times. Cellular extracts were assayed for PDP kinase
activity using a proline-rich peptide as substrate. Shown are the mean
values (±S.D.) of three experiments performed in triplicates.
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Fig. 5.
Inhibition of TNF receptor internalization
prevents TNF stimulation of JNK. U937 cells were left untreated
(a and d), subjected to MDC treatment
(b and e), or K+-depleted
(c and f) before stimulation with either 100 ng/ml TNF for indicated times (a, b, and
c) or 100 µg/ml C6-ceramide for 30 min
(d, e, and f). JNK was
immunoprecipitated from cellular lysates, and JNK activity was assessed
using a GST-c-Jun-1-135 fusion protein as substrate. Arrows
indicate the phosphorylated 34-kDa GST-c-Jun fusion protein. Results
are representative of three experiments. The relative amounts of
GST-c-Jun substrate phosphorylation in response to TNF (g)
and to C6-ceramide (h) were determined by
two-dimensional laser densitometry of the respective autoradiographs
(a, b, and c) and (d,
e, and f). , control; , MDC; ,
K+ depletion.
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MDC Does Not Block the Association of FAN and TRADD with
TR55--
We next examined whether the differential inhibitory effects
of MDC on TNF receptor death domain signaling could be explained by
selective interference with the association of death domain-binding protein TRADD and the adaptor protein FAN to TR55. For this purpose, HEK 239 cells were transfected with the FLAG-tagged full-length FAN or
TRADD fusion constructs, and cellular lysates were subjected to
immunoprecipitation using the anti-TR55 antibody htr-9. The co-immunoprecipitating FAN and TRADD-proteins were detected by Western
blotting using anti-FLAG antibody M5. As shown in Fig. 6, MDC treatment did not reduce the
association of FAN or TRADD with TR55. Thus MDC does not seem to
selectively affect the stability of TR55·TRADD complexes, indicating
that the specific action of MDC on TR55 death domain signaling cannot
be explained at the level of TR55-associated proteins.

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Fig. 6.
MDC does not inhibit association of FAN or
TRADD with TR55. HEK 293 cells were transiently transfected with
20 µg of either pFLAG.CMV2-FAN (A) or pRK5.FLAG-TRADD
(B) and cultured for 18 h. Cells were left untreated or
were treated with 100 µg/ml MDC for 1 h. Cellular lysates were
prepared and directly applied to SDS-polyacrylamide gel electrophoresis
for control of FAN and TRADD expression. Immunoprecipitation
(IP) was performed using the anti-TR55 antibody htr-9. The
presence of FAN and TRADD was analyzed by Western blotting using
anti-FLAG antibody M5 and the ECL immunodetection system.
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TNF-mediated Cell Death Is Blocked by MDC--
The induction of
programmed cell death is one important hallmark of TNF, CD95 ligand as
well as ceramide action. To study the effects of the inhibition of
receptor internalization on cell death, we performed pretreatment of
cells with MDC, which did not cause significant toxic side effects when
applied at prolonged times. In contrast, K+ depletion of
cells for 24 h proved to be toxic for U937 cells (data not shown)
and, therefore, could not be employed to study long term effects of TNF
such as the induction of cell death.
As shown in Fig. 7, TNF and exogenous
C6-ceramide induced the death of U937 cells after 24 h. TNF-mediated cytotoxicity was prevented by MDC pretreatment in a
dose-dependent manner, with half-maximal protection between
25 and 50 µM MDC, corresponding to the
dose-dependent MDC inhibition of TNF receptor
internalization (Fig. 1B). At 100 µM MDC, the
fraction of TNF-induced dead cells estimated by MTT assays was reduced
from 63 to 24%, corresponding to a 62% inhibition (Fig. 7). In
contrast, the cytotoxic effect of exogenous C6-ceramide
taken up by the cells by pinocytosis was not affected by MDC. The
protective effect of MDC on TNF-induced cytotoxicity was observed up to
48 h after TR55 triggering (43.5% inhibition at 100 µM MDC, data not shown), indicating that inhibition of
TNF receptor internalization by MDC provides significant long term
protection against TNF-induced cell death.

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Fig. 7.
TNF-mediated cytotoxicity is blocked by MDC
and can be reconstituted by exogenous ceramide. U937 cells were
left untreated (C, open bars) or incubated with
either 30 ng/ml TNF (black bars) or 25 µM
C6-ceramide (Cer, gray bars) after
pretreatment with the indicated concentrations of MDC. Cell viability
was assessed by MTT assays after 24 h. The values represent the
mean of a representative experiment performed in quadruplicates
(±S.D.).
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In dose-response experiments, we evaluated whether pretreatment with
MDC was effective in inhibiting death of U937 cells at different
concentrations of TNF, ceramide, and anti-CD95 antibody. Fig.
8 shows the dose-dependent
cytotoxicity induced by TNF, ceramide, and CD95 triggering. MDC was
protective against TNF-induced cell death (Fig. 8A), whereas
ceramide- and CD95-induced cell death were not affected by MDC (Fig. 8,
B and C). These results suggest that the
protective effect of MDC appears to be selective for TNF-mediated cell
death.

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Fig. 8.
TNF, ceramide, and anti-CD95
antibody-mediated cytotoxicity is differentially affected by MDC.
U937 cells were left untreated or were pretreated with 100 µM MDC for 1 h. Aliquots of control or MDC treated
cells were incubated with indicated concentrations of TNF
(A), C6-ceramide (B), or agonistic
anti-CD95-antibody (C). Cell viability was assayed by MTT
assays after 24 h as in Fig. 7. The values represent the mean
values of three experiments performed in quadruplicates (±S.D.).
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In morphological analyses, TNF-induced chromatin condensation in U937
cells, typical for apoptosis, is shown (Fig.
9b). Incubation with 100 µM MDC before TNF treatment significantly reduced the fraction of apoptotic cells from 65 to 19%, corresponding to a 70.8%
inhibition of TNF-induced apoptosis (Fig. 9, f and
i). Apoptosis was also induced by exogenous
C6-ceramide (Fig. 9c) that, again, was not
inhibitable by MDC (Fig. 9, g and i). The
protective effect of MDC against TNF-induced apoptosis was also
observed in a different cell line, L929, indicating that the role of
TNF receptor internalization for signaling cell death is not restricted
to U937 cells (data not shown). As demonstrated in Fig. 9
(d, h, and i), induction of apoptosis
in Jurkat cells by anti-CD95 antibody could not be blocked by MDC
pretreatment. Similar results were obtained by terminal
deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL)
staining for apoptosis (data not shown). Together, these findings
suggest that the anti-apoptotic effect of MDC observed in TNF-treated
cells is specific for cell death mediated by TR55.

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Fig. 9.
MDC selectively inhibits TNF-induced
apoptosis. U937 cells were left untreated (a-d) or
were treated with 100 µM MDC for 24 h.
(e-h). Aliquots were stimulated with either 30 ng/ml TNF
(b and f), 25 µM
C6-ceramide (c and g), or 30 ng/ml
anti-CD95 (d and h). After 24 h, the cells
were stained with acridine orange and ethidium bromide. The percentage
of apoptotic cells was determined by counting a minimum of 300 cells
(i).
|
|
 |
DISCUSSION |
In the present paper the role of receptor internalization for TNF
signaling was investigated. Intriguingly, inhibition of TR55
internalization by MDC or K+ depletion abolished select
signaling events while leaving others unaffected. In particular,
TNF-induced activation of A-SMase, JNK, and the induction of apoptosis
were sensitive to MDC. In contrast, MDC did not inhibit TNF-induced
activation of N-SMase and PDP kinases. Similar results were obtained
with K+-depletion, which inhibited TNF receptor
internalization and, like MDC, selectively blocked TNF-induced
activation of A-SMase and JNK but not N-SMase or PDP kinases.
MDC has been extensively used to block endocytosis and trafficking of
various ligand-receptor systems. MDC is known as a potent competitive
inhibitor of transglutaminase (36, 37, 58, 59). The enzymatic action of
transglutaminase involves cross-linking of proteins by forming an
isopeptide bond between a lysine residue of one protein and a glutamine
residue of another protein during coated pit formation. MDC has been
shown to block endocytosis of
2-macroglobulin and many
polypeptide hormones and cytokines like epidermal growth factor,
hepatocyte growth factor, insulin-like growth factor-I, and IL-8 (37,
38, 40, 43, 44, 58, 60). MDC-mediated inhibition of transglutaminase
has also been implicated in the blocking of endocytosis of vesicular
stomatitis virus, Semliki Forest virus, and other types of endocytosis
occurring through clathrin-coated vesicles (61, 62). MDC does not
affect the number or affinity of TNF receptors expressed on the cell surface (41).2
MDC blocked TNF receptor internalization at concentrations similar to
the concentrations required for inhibition of transglutaminase in
lysates from Chinese hamster ovary cells (37), suggesting that the
inhibition of this enzyme is related to the MDC effects on TNF receptor
endocytosis. MDC concentrations exceeding 100 µM did not
further increase the inhibitory effect on TNF receptor internalization
in U937 cells. These findings are consistent with a previous study by
Davies and co-workers, reporting on MDC-mediated inhibition of
2-macroglobulin receptor clustering (37).
The differential requirement of TR55 internalization for the activation
of A-SMase and N-SMase are in concordance with a previous report by
Hofmeister et al. in which the authors used an independent approach to study the functional consequences of IL-1 receptor internalization (63). An IL-1 receptor type I-positive EL4 thymoma cell
line was employed, which is defective in the IL-1R accessory protein
(IL-1RAcP) required for IL-1 receptor internalization. In this IL-1R
internalization-defective cell line, IL-1 induction of N-SMase appeared
normal, whereas the activation of A-SMase was completely impaired.
Transfection with IL-1RAcP cDNA restored IL-1 receptor
internalization as well as IL-1-induced A-SMase stimulation, indicating
the requirement of IL-1R internalization for A-SMase activation.
Our findings on the differential requirement of receptor
internalization for activation of A- and N-SMase may help to explain previous data by Andrieu et al. (51). These authors could
not detect a requirement of TNF or IL-1 receptor internalization for sphingomyelin turnover. Here we show that it is only N-SMase that is
insensitive to MDC or K+ depletion, whereas activation of
A-SMase depends on TNF receptor internalization. Because the overall
enzymatic activity of A-SMase exceeds that of N-SMase by a factors of
3-10, depending on the cell line investigated (Fig. 4 and Refs. 9 and
65), the remaining N-SMase activity might have escaped the authors'
detection system.
A role for receptor clustering and internalization for activation of
JNK has been also suggested by Rosette and Karin (64), who report that
exposure of HeLa cells to UV light or osmotic shock induced clustering
and internalization of cell surface receptors for TNF, epidermal growth
factor, and IL-1 in the absence of the respective ligands. UV or
osmotic shock-induced activation of JNK could be inhibited by blocking
receptor clustering at 10 °C or after receptor down-regulation,
suggesting that multimerization and clustering of the cytokine
receptors is the initial signaling event that activates the JNK cascade.
Intriguingly, MDC blocked TR55-induced apoptosis. The doses of MDC
required to inhibit TNF receptor endocytosis correlated well with those
needed for the inhibition of TNF-induced cytotoxicity, consistent with
the idea that TR55 internalization is required for apoptotic signaling.
It is worth mentioning that MDC does not prevent apoptosis in a
nonspecific manner. C6-ceramide, which is taken up through
pinocytosis, triggered programmed cell death in the presence of MDC. In
addition, CD95-mediated apoptosis of U937 and Jurkat cells was not
prevented by MDC, suggesting that CD95-induced cell death is regulated
by a mechanism distinct from clathrin-coated pit-mediated endocytosis.
Because the effects of MDC inhibition are presumably based on a
blockade of protein cross-linking mediated by transglutaminase, the
question is raised of whether MDC might disrupt the interaction of
TRADD with the TR55 death domain. This would result in an uncoupling of
death domain signaling independent of TR55 internalization. Our
results, however, clearly show that association of TRADD to the death
domain of TR55 was not impaired by MDC, suggesting that inhibition of
A-SMase, JNK activation, and apoptosis by MDC does not result from
disrupting the TRADD/TR55 signaling complex.
The importance of TNF receptor internalization for signaling cell death
is controversial. A functional role of TNF receptor internalization for
mediating TNF cytotoxicity has been suggested previously by Kull and
Cuatrecasas (55), Watanabe et al. (56), and Pastorino
et al. (32). It should be emphasized that a membrane-bound form (26 kDa) of TNF triggers cell death by direct cell-to-cell contact
and, indeed, is apparently independent of TNF receptor internalization
(57, 66). It is important to note, however, that membrane-bound TNF has
been shown to preferentially trigger the p75 TNF receptor (TR75) (57).
TNF binding to TR75 does not result in receptor internalization but
rather triggers shedding of the TR75 (49, 50). Clearly, the mechanism
by which TR75 signals cytotoxicity is different from the TR55 signaling
pathway. Unlike membrane-bound TNF, soluble TNF binds and triggers TR55 that is readily internalized following ligand binding. Thus, the conflicting results can be explained by the engagement of distinct TNF
receptors by membrane-bound and -soluble TNF, respectively.
Our study implies that the functional consequences of TR55 endocytosis
go beyond receptor degradation to terminate ligand-induced signaling.
The results of our study indicate that TR55 internalization is
crucially important for targeting and coupling of the TR55 adapter
protein complexes to specific signaling systems like A-SMase, JNK, and
a proapoptotic signaling cascade. TNF receptor endocytosis may play a
previously unrecognized role in relaying TNF signals to intracellular
compartments, where TNF receptor bearing membranes can be sensed as
"signaling rafts."