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Departments of
*
Medicine and
Clinical Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom; and
Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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55 kDa) and TNF-R2 (75 kDa). TNF binding is followed by
receptor clustering and internalization of the TNFR complex by
endocytosis (4). However, the role of ligand or receptor
internalization in the induction of cellular responses remains unclear.
Inhibition of receptor internalization reduces some aspects of TNF
signaling (5, 6), but it is unlikely that the ligand needs to be
internalized because many effects of TNF can be mimicked by agonistic
antireceptor Abs (7, 8, 9).
Several proteins couple TNFRs to signaling cascades through interaction
with the intracellular domains of TNF-R1 and TNF-R2. TNF-R1 interacts
in a ligand-dependent process with a 34-kDa TNFR-associated death
domain protein (TRADD)3
leading to the activation of NF-
B, Jun N-terminal, and p38 activated
protein kinases and programmed cell death (10, 11). In contrast, TNF-R2
has been shown to interact with heterocomplexes of TNFR-associated
factors 1 and 2 (TRAF1 and TRAF2) in a non-TNF-dependent manner (12).
TNF-R1 and TNF-R2 are traditionally regarded as cell surface receptors, although we have reported that TNF-R1 is primarily a Golgi-associated protein (13) with only a minor subpopulation of receptors expressed in the plasma membrane. However, the precise Golgi subcompartment (cis-, medial-, trans-Golgi cisternae) or the trans-Golgi network (TGN) in which TNF-R1 resides had not been characterized. With respect to TNF-signaling pathways, this may be important since the TGN is responsible not only for sorting proteins, endosomes and lysosomes, to the cell surface (14, 15, 16, 17, 18), but also for retrieving and reusing plasma membrane components internalized by endocytosis (19, 20, 21). In this study, we have defined the precise Golgi subcompartment in which TNF-R1 is localized and determined the potential of TRADD to associate with TNF-R1 in the Golgi compartment after TNF treatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Medium 199, RPMI 1640 medium, FCS, L-glutamine, penicillin/streptomycin, trypsin-EDTA, brefeldin-A (BFA), sucrose, Trisma-Base, NaCl, EDTA, Triton X-100, aprotinin, PMSF, leupeptin, and pepstatin A were all purchased from Sigma-Aldrich (Poole, U.K.). Human rTNF was provided by Autogen Bioclear (P30001A; Potterne, U.K.). The mouse monoclonal anti-human TNF-R1 was obtained from Genzyme Diagnostic (1995-01; Cambridge, MA), goat polyclonal anti-human TNF-R1 Abs were from R&D Systems (AB-225-PB; Minneapolis, MN), mouse monoclonal anti-p58 was from Sigma (G2404; St. Louis, MO) and mouse monoclonal anti-TRADD Ab was from Transduction Laboratories (T50320; Lexington, KY). The rabbit polyclonal Ab to TGN46 was a kind gift from Dr. S. Ponnambalam at the University of Dundee. Secondary FITC-conjugated Abs were from Sigma and Texas Red-conjugated Abs from Dako (Carpinteria, CA). Horseradish peroxidase (HRP) -conjugated donkey anti-mouse IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA) and HRP-conjugated porcine anti-rabbit from Dako. Protein G Sepharose 4 fast flow was from Pharmacia (Piscataway, NJ).
Cell culture
ECV304 cells, a spontaneously immortalized cell line of human umbilical vein origin (22) (a kind gift from I. Fritz, Babraham Institute, Cambridge, U.K.) were cultured in medium 199 supplemented with 10% FCS, L-glutamine, and antibiotics. U937 cells, a human monocyte cell line, were cultured in RPMI 1640 medium supplemented with 10% FCS, L-glutamine, and antibiotics.
Immunofluorescence microscopy
ECV304 cells were seeded onto sterile glass coverslips in 24-well plates (Costar, High Wycombe, U.K.) 16–24 h before fixation. Cells were washed in PBS, fixed in 2% paraformaldehyde for 2 min, and permeabilized with 0.1% Triton X-100 for 2 min. U937 cells were washed in PBS, fixed as above, cytospun at 600 rpm for 15 s, and permeabilized as above. Cells were incubated with primary Abs diluted in 1% BSA/PBS for 1 h at room temperature. After they were washed, the cells were incubated with secondary conjugated Abs for 1 h at room temperature. Coverslips or slides were mounted in Citifluor and examined using a Nikon Optiphot-II microscope (Nikon, Kingston upon Thames, U.K.) coupled to a Bio-Rad (MRC1000; Hemel Hempstead, U.K.) confocal attachment and COMOS software (BioRad).
Transfection of human TNF-R1 and TNF-R2 into bovine aortic endothelial cells (BAEC)
Expression constructs encoding the TNFR (TNF-R1, pcDNA3, and TNF-R2, pcDNA3) were generated by ligating NotI fragments containing the coding sequences from an expression vector generously provided by Immunex (Seattle, WA) into NotI-digested pcDNA3.1 (Invitrogen, San Diego, CA). BAEC were cultured in DMEM supplemented with 10% FCS, 5 mM HEPES, L-glutamine (1:100) sodium phosphate, penicillin, and streptomycin (all from Life Technologies, Gaithersburg, MD). Subconfluent BAEC in 35 mm tissue culture dishes (C-6) were transfected with the TNFR expression constructs (3 mg/0.2 ml Optimen with 6 ml lipofectamine/0.2 ml according to the manufacturer’s directions (Life Technologies). After culture overnight, cells were transferred to 10-cm-plates and transfectants were selected with 0.5 mg ml-1 G418 (Life Technologies).
Subcellular fractionation
ECV304 cells (detached using PBS containing 0.05% EDTA) and
U937 cells were washed three times in PBS (600 x g
for 5 min). All subsequent steps were performed at 4°C. The washed
cell pellets were resuspended (1 x 108 cells
ml-1) in homogenization buffer (10 mM Tris-HCl (pH 7.4),
containing 3% (w/v) sucrose and proteinase inhibitors - 1 mM
EDTA, 10 µg ml-1 aprotinin, 1 µg ml-1
leupeptin, 1 µg ml-1 pepstatin A and 1 mM PMSF) and
allowed to swell in the hypotonic buffer for 20 min. The cells were
then homogenized in buffer containing 0.25 M sucrose using 20–30
strokes in a Dounce homogenizer. The homogenate was centrifuged
(1300 x g for 5 min) to obtain a nuclear-cell
debris pellet and postnuclear supernatant (PNS). The PNS was further
centrifuged (10,000 x g for 15 min) and the
postmitochondrial/lysosomal supernatant (10K supernatant) was further
centrifuged (100,000 x g for 1 h) to yield a
cytosolic fraction (100K supernatant) and a microsomal pellet (100K
pellet). The 100K pellet was resuspended in 1.5 M sucrose and overlaid
with 1.15 M, 0.9 M, 0.6 M, and 0.25 M sucrose. The discontinuous
sucrose gradient was centrifuged at 100,000 x g
for 2 h. The individual interfaces were removed, diluted in 0.25 M
sucrose, and centrifuged (100,000 x g for 1
h). The pellets were resuspended in 500 µl of 0.25 M sucrose and
assayed for alkaline phosphodiesterase activity (plasma membrane) and
mannosidase II activity (Golgi marker) as described by Storrie and
Madden (23) and the protein concentrations were determined using the
Bradford assay (24). Samples were used immediately or stored at
-20°C.
SDS-PAGE and Western blot analysis
Equivalent amounts of fractionated protein (5–25 µg) were resolved by 10% SDS-PAGE and blotted onto nitrocellulose membranes. Blots were blocked in buffer containing Tris-HCl (pH 7.4), 5% dried milk powder, and 0.05% Tween 20. After blocking, the membrane was immunoblotted with mouse monoclonal anti-TNF-R1 (1:1500), mouse monoclonal anti-p58 (1:500), mouse monoclonal anti-TRADD (1:500), or rabbit anti-TGN46 (1:5000) for 2 h at room temperature or overnight at 4°C. The bound Abs were visualised by chemiluminescence (Kirkegaard & Perry Laboratory, Gaithersburg, MD).
Coimmunoprecipitation
U937 cells (5 x 107 ml-1) were washed three times in PBS (600 x g for 5 min) and incubated in the presence or absence of TNF (100 ngml-1) for variable time periods. The cells were washed once in PBS (600 x g for 5 min) and either lysed immediately in 500 µl of lysis buffer (20 mM Tris (pH 7.5), 100 mM NaCl, 1% Triton X-100, and proteinase inhibitors) for 1 h on ice or chased for variable time periods in PBS then lysed or to study the effect of inhibiting receptor mediated endocytosis, the cells were chased in prewarmed PBS or 0.45 M sucrose for variable time periods before lysis. In the meantime, prewashed Protein G Sepharose beads (40 µl) were incubated with 20 µl of goat anti-TNF-R1 Ab in 500 µl of lysis buffer using an upright rotator (1 h, 4°C). The U937 lysate was centrifuged (10,000 x g for 15 min at 4°C) and the supernatant incubated with the Protein G/Ab complex for 3 h at 4°C. The Sepharose beads were then washed four times with lysis buffer and twice with lysis buffer lacking Triton X-100. The precipitates were resuspended in nonreducing Laemmli sample buffer, boiled, resolved by SDS-PAGE, and transferred onto nitrocellulose. After blocking, the membrane was immunoblotted with the mouse monoclonal anti-TRADD Ab (1:500) for either 2 h at room temperature or overnight at 4°C and visualized with HRP-conjugated donkey anti-mouse IgG using the chemiluminescence detection system.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our previous report (13), we showed that in HUVEC, TNF-R1 is
principally localized in the Golgi apparatus. Although several
different Abs revealed Golgi staining, not all Abs reacted with all
cells. Therefore, we transfected expression plasmids for TNF-R1 and
TNF-R2 to confirm that there was specific recognition of TNF-R1 in the
Golgi. BAECs transfected with human TNF-R1 exhibited a compact
juxtanuclear staining pattern (Fig. 1
A) characteristic of
Golgi-associated proteins (25) when stained with monoclonal
anti-human TNF-R1 Ab (R&D Systems). This Ab did not stain
untransfected BAEC. In contrast, BAEC transfected with human TNF-R2
demonstrated diffuse cytoplasmic and surface membrane staining with
anti-human TNF-R2 Abs (Fig. 1B). Because untransfected
BAECs do not stain with the Ab to human TNF-R1, the Golgi-associated
staining can be unequivocally attributed to TNF-R1 localization to this
subcellular compartment.
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). Both ECV304 and U937
cells exhibit a staining pattern characteristic of Golgi-associated
proteins when labeled with a mouse mAb to TNF-R1 (Figs. 3 and 4).
We extended this morphological approach by immunoblotting TNF-R1 in
fractionated cells. In both ECV304 (Fig. 2, A and
B) and U937 cells (Fig. 2C) TNF-R1 was enriched
in the 0.9/1.15 M interface together with p58, a
medial-Golgi marker, and TGN46, a trans-Golgi
marker. In Fig. 2B, the TGN46 polyclonal Ab recognizes a set
of closely spaced bands of 100–120 kDa attributable to TGN46 being
highly glycosylated (26). mannosidase II specific activity was
7-fold higher in this interface than the PNS. The enrichment of TNF-R1
in this Golgi-containing fraction provides biochemical evidence for the
Golgi association of TNF-R1, complementing the previous
immunocytochemical approaches.
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To investigate with which Golgi subcompartment
(cis-, medial-, trans-Golgi cisternae
or TGN) the receptor is associated, cells were cultured in the presence
or absence of BFA and again analyzed by confocal microscopy. This
fungal metabolite causes the different Golgi compartments to
redistribute in a characteristic manner. In many cell types, the TGN
collapses around the microtubule organizing center (MTOC) giving a
spotlike appearance (27), whereas other Golgi compartments redistribute
into the endoplasmic reticulum (ER) giving a cytoplasmic staining
pattern (28). After treating ECV304 cells for 4 h with BFA, the
immunofluorescence pattern associated with TNF-R1 and TGN46 was that of
a compact center of staining at the nuclear membrane, representative of
a collapse to the MTOC, whereas the immunofluorescence pattern
associated with p58 was now dispersed throughout the cytoplasm
consistent with the redistribution of p58 into the ER (Fig. 3
). In all
cases, the collapse to the MTOC and the redistribution to the ER was
rapid, occurring within 10 min. Merged images following costaining with
the rabbit polyclonal anti-TGN46 Ab and the mouse monoclonal
anti-TNF-R1 Ab demonstrated colocalization (Fig. 4), although
anti-TNF-R1 Abs additionally exhibited weak cytoplasmic staining.
Identical staining patterns were also observed in U937 cells (Fig. 5). We conclude that most TNF-R1 resides
in the TGN, but smaller receptor pools also exist.
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The principal signal transduction pathways initiated by ligand
binding to TNF-R1 are mediated by the recruitment of TRADD. To
determine whether the TGN was involved in TNF signaling, we
investigated whether TRADD also demonstrated a Golgi distribution. By
indirect immunofluorescence using U937 cells (Fig. 5) and ECV304 cells
(data not shown), the monoclonal anti-TRADD Ab exhibited a compact
perinuclear stain characteristic of Golgi-associated proteins and
similar to that observed with anti-TNF-R1 Abs on cells cultured
both in the presence and absence of TNF. After BFA treatment, the
immunofluorescence pattern associated with anti-TRADD Abs dispersed
throughout the cytoplasm (Fig. 5) suggesting redistribution to the ER.
This pattern suggests a medial- or cis-Golgi
association, unrelated to the distribution of TNF-R1. In contrast to
the staining pattern, Western blot analysis of subcellular fractions
derived from cells cultured in the absence of TNF suggested that TRADD
was a cytosolic protein (Fig. 6A), whereas under the same
conditions TNF-R1 was associated with membrane containing fractions
(Fig. 6B). These data suggest that in untreated cells, TRADD
is loosely associated with the Golgi but not the TGN in which TNF-R1 is
localized.
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In U937 cells, TRADD associates with TNF-R1 in a TNF-dependent
process (11). Therefore, we used U937 cells to examine whether TRADD
could be recruited to TNF-R1 in the Golgi. First, we examined the
ability to coimmunoprecipitate TRADD with TNF-R1. TRADD
coimmunoprecipitated with TNF-R1 in TNF-treated, but not mock-treated,
cells from the whole cell lysate and 10K pellet, but poorly from the
100K membrane pellet that contains TGN-derived membranes (Fig. 7). Because plasma membranes are found to
a greater extent in the 10K pellet, these results are consistent with
the conclusion that TRADD coimmunoprecipitated in the 10K pellet is
associated with plasma membrane TNF-R1. The failure to find TRADD in
immunoprecipitates from the 100K membrane pellet of TNF-treated cells,
suggests that the Golgi complex is not involved in signaling in the
response to exogenous TNF.
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To examine this possibility the kinetics of TNF-dependent TRADD
coimmunoprecipitation at both 4°C (inhibits receptor mediated
endocytosis) and room temperature was determined. The recruitment of
TRADD to TNF-R1 in U937 cells is rapid, occurring within 1 min even at
4°C (Fig. 8A). Therefore, we
pulsed U937 cells with TNF for 1 min at 4°C and chased in PBS for 1
or 60 min at 20°C, prepared the 10K and 100K pellets, and examined
the ability to coimmunoprecipitate TRADD from each of these subcellular
fractions. TRADD was coimmunoprecipitated from the 10K pellet after 1
min, but not after 60 min, whereas TRADD was not coimmunoprecipitated
from the 100K pellet at either time point (Fig. 8B). These
data are consistent with the conclusion that TRADD blotted in the 10K
pellet was attributable to the presence of plasma membrane TNF-R1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have studied the localization of TNF-R1 in different cell types with diverse biologic responses to TNF. Cultured human endothelial cells are resistant to TNF-induced cell death, but respond to this cytokine by activating a number of proinflammatory genes (30). In other cell types, TNF is cytotoxic and U937 cells are reported to be sensitive to TNF-induced apoptosis (31). As TNF-R1 is predominantly localized in the Golgi of both endothelial cells and U937 cells, this cellular distribution is unlikely to be important in determining these different responses to TNF.
Our findings indicate that in U937 cells exogenous, TNF causes TRADD to rapidly associate with TNF-R1 at the plasma membrane. It is believed that trimeric TNF induces trimeric clusters of TNF-R1 and the death domains of the clustered receptor create a high affinity binding site for TRADD (11). Our studies demonstrate that subsequent internalization of the TNF-TNF-R1 complex results in the dissociation of TRADD. This could be attributed to ligand dissociation when the endocytosed receptor-ligand complex comes into contact with the acidic environment of endosomes. When TNF is released from its receptor, the receptor clusters dissociate causing TRADD to be released. As most of the signaling events are completed within minutes of the exposure of cells to TNF, the inability to coimmunoprecipitate TRADD from Golgi-enriched subcellular fractions (100K pellet) within 30 min suggests that exogenous TNF does not induce TRADD recruitment to the Golgi. Our observations do not exclude the possibility that endogenously synthesized TNF may interact with the Golgi-associated TNF-R1. Expression of a transfected-TNF gene in TNF-sensitive tumor cell lines confers resistance to TNF-mediated cell lysis (32), which is correlated with down-modulation of cell surface TNFR (33). Secreted TNF is processed through the Golgi and released by a BFA-sensitive mechanism (34). Further studies will determine whether endogenous TNF can modify cellular response to TNF through interaction with a Golgi receptor. Interestingly, the indirect immunofluorescence studies demonstrated that, in cells which had not been treated with TNF, TRADD was associated with a different Golgi subcompartment to TNF-R1. The mechanism by which the cytosolic protein TRADD associates with the Golgi is unknown, but their different localizations within the Golgi may prevent constitutively synthesised TRADD and TNF-R1 interacting.
The significance of the TGN association of TNF-R1 remains unknown. The TGN packages newly synthesized proteins into transport vesicles that are directed to pre-lysosomal/lysosomal compartments, secretory granules, or the plasma membrane (14, 15, 16, 17) and it participates in retrieving and reusing plasma membrane components internalized by endocytosis such as the mannose-6-phosphate and transferrin receptors (35, 36). As TNF-R1 after TNF binding is also rapidly endocytosed, the internalized receptor ligand complex may recycle to the TGN or other intracellular compartments and act locally to generate TNF-driven signals. Alternatively, the TGN-associated receptor may act as a pool from which the receptor is delivered in a regulated manner to other sites.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John Bradley, Department of Medicine, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, U.K. E-mail address:
3 Abbreviations used in this paper: TRADD, TNFR-associated death domain protein; TRAF, TNFR-associated factors; TGN, trans-Golgi network; HRP, horseradish peroxidase; BAEC, bovine aortic endothelial cells; BFA, brefeldin A; PNS, post nuclear supernatant; MTOC, microtubule organizing center; ER; endoplasmic reticulum.
Received for publication June 23, 1998. Accepted for publication September 30, 1998.
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B activation. J. Biol. Chem. 41:24518.
B activation. Cell 81:495.[Medline]
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M. Mancini, C. E. Machamer, S. Roy, D. W. Nicholson, N. A. Thornberry, L. A. Casciola-Rosen, and A. Rosen Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis J. Cell Biol., May 1, 2000; 149(3): 603 - 612. [Abstract] [Full Text] [PDF]
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X. D. Zhang, A. V. Franco, T. Nguyen, C. P. Gray, and P. Hersey Differential Localization and Regulation of Death and Decoy Receptors for TNF-Related Apoptosis-Inducing Ligand (TRAIL) in Human Melanoma Cells J. Immunol., April 15, 2000; 164(8): 3961 - 3970. [Abstract] [Full Text] [PDF]
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X. Feng, M. L. Gaeta, L. A. Madge, J.-H. Yang, J. R. Bradley, and J. S. Pober Caveolin-1 Associates with TRAF2 to Form a Complex That Is Recruited to Tumor Necrosis Factor Receptors J. Biol. Chem., March 9, 2001; 276(11): 8341 - 8349. [Abstract] [Full Text] [PDF]
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E. Boone, T. Vanden Berghe, G. Van Loo, G. De Wilde, N. De Wael, D. Vercammen, W. Fiers, G. Haegeman, and P. Vandenabeele Structure/Function Analysis of p55 Tumor Necrosis Factor Receptor and Fas-associated Death Domain. EFFECT ON NECROSIS IN L929sA CELLS J. Biol. Chem., November 22, 2000; 275(48): 37596 - 37603. [Abstract] [Full Text] [PDF]
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V. Cottin, A. A. Van Linden, and D. W. H. Riches Phosphorylation of the Tumor Necrosis Factor Receptor CD120a (p55) Recruits Bcl-2 and Protects against Apoptosis J. Biol. Chem., May 11, 2001; 276(20): 17252 - 17260. [Abstract] [Full Text] [PDF]
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Y. Jin, S. J. Atkinson, J. A. Marrs, and P. J. Gallagher Myosin II Light Chain Phosphorylation Regulates Membrane Localization and Apoptotic Signaling of Tumor Necrosis Factor Receptor-1 J. Biol. Chem., August 3, 2001; 276(32): 30342 - 30349. [Abstract] [Full Text] [PDF]
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C. De Nadai, P. Sestili, O. Cantoni, J.-P. Lievremont, C. Sciorati, R. Barsacchi, S. Moncada, J. Meldolesi, and E. Clementi Nitric oxide inhibits tumor necrosis factor-alpha -induced apoptosis by reducing the generation of ceramide PNAS, May 9, 2000; 97(10): 5480 - 5485. [Abstract] [Full Text] [PDF]
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H.-B. Shu and H. Johnson B cell maturation protein is a receptor for the tumor necrosis factor family member TALL-1 PNAS, August 1, 2000; 97(16): 9156 - 9161. [Abstract] [Full Text] [PDF]
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