©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cross-linking of the p55 Tumor Necrosis Factor Receptor Cytoplasmic Domain by a Dimeric Ligand Induces Nuclear Factor-B and Mediates Cell Death (*)

(Received for publication, March 3, 1995)

Dieter Adam(§)(¶) , Ulrike Keler , Martin Krönke (§)

From the Institut für Medizinische Mikrobiologie und Hygiene, Technische Universität München, Trogerstrasse 32, 81675 München, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have fused the cytoplasmic domain of the p55 tumor necrosis factor (TNF) receptor to the extracellular and transmembrane domain of the mouse platelet-derived growth factor (PDGF) receptor. Mouse mammary gland epithelial (NMuMG) cells were stably transfected with the PDGFR-TR55 chimeric receptor. These cells lack endogenous PDGF receptor expression and do not respond to PDGF. In the PDGFR-TR55 transfectants, PDGF elicited a cytotoxic response, which is indistinguishable from that induced by the wild type p55 TNF receptor. In addition, PDGF-induced activation of the PDGFR-TR55 chimeric receptor resulted in nuclear translocation of NF-B. The data presented suggest that cross-linking of the p55 TNF receptor cytoplasmic domain by a dimeric ligand such as PDGF is sufficient to generate cellular responses that do not differ from those observed with the trimeric ligand TNF.


INTRODUCTION

Membrane receptors for TNF()signal a large number of cellular responses, including immunoregulatory activities, anti-viral activity, cytotoxicity, and the transcriptional regulation of many genes(1, 2, 3, 4) . Two distinct cell surface molecules of 55 kDa (TR55) and 75 kDa (TR75) apparent molecular mass have been cloned(5, 6, 7, 8, 9, 10) . Numerous studies have demonstrated that the majority of TNF activities is mediated by TR55(11, 12, 13) . Signal transduction through TR55 involves at least two independent pathways, one initiated by a neutral sphingomyelinase, which results in subsequent activation of a ceramide-activated protein kinase, phospholipase A, as well as the protein kinase Raf(14, 15, 16) . The other pathway involves activation of a phosphatidylcholine-specific phospholipase C, which regulates stimulation of protein kinase C and an acidic sphingomyelinase, which in turn mediates induction of the transcription factor NF-B(15, 17) . NF-B controls expression of a variety of TNF-responsive genes (18, 19, 20) and, among other molecules, represents an important mediator of the proinflammatory responses caused by TNF.

Most cell surface receptors studied, e.g. tyrosine kinase receptors like the PDGF receptor, are activated by ligand-induced dimerization of the receptor subunits (for reviews, see Refs. 21 and 22). Unlike these receptors, TR55 (and probably most other members of the TNF receptor superfamily) can form a trimeric structure induced by binding of a trimeric ligand(23, 24, 25) . However, the trimeric structure of TR55 observed in x-ray diffraction studies may represent only one of several possible conformations in intact cells(26) .

To determine whether trimer formation of the p55 TNF receptor is absolutely required for signaling functions, the extracellular and transmembrane domain of the TR55 was replaced by domains of an unrelated molecule that is not activated by a trimeric ligand. The PDGFR was chosen because it represents one of the best characterized receptor tyrosine kinases(22, 27) . In addition, it has clearly been demonstrated that the homo-dimeric structure of the receptor ligand (in this case PDGF-BB) activates the receptor by dimerizing two subunits(28, 29) .

Here, we demonstrate that PDGF binds to the PDGFR-TR55 chimeric receptor stably expressed in NMuMG cells and induces a cytotoxic response in these transfectants as well as nuclear translocation of NF-B. These results support the concept that dimerization of TR55 is sufficient to mediate TNF-specific responses.


MATERIALS AND METHODS

Plasmids and Reagents

The expression vectors pADB-TR55, containing the full-length human p55 TNF receptor cDNA, pAD-CMV1, and pSVRCD4 have been described(6, 11, 31) . Murine and human recombinant TNF- were provided by Dr. G. Adolf (Boehringer Ingelheim, Vienna), and human recombinant BB-chain PDGF was obtained from Drs. B. Ratzkin and L. Souza (Amgen, Thousand Oaks). Antibody SM14 was kindly provided by Dr. J. B. Bolen (Bristol-Myers Squibb, Princeton). Anti-PDGF receptor monoclonal antibodies were purchased from Affinity Research Products (Nottingham).

Construction of the PDGFR-TR55 Molecule

The extracellular domain and parts of the cytoplasmic domain of TR55 were removed from the expression vector pADB-TR55 by digestion with SalI/HindIII. The extracellular and transmembrane domain of the murine PDGF receptor was recovered from the expression vector pSVRCD4 as an EcoRI-HindIII DNA fragment and ligated into the SalI-HindIII site of pADB-TR55 after treatment with Klenow polymerase to restore the reading frame. The resulting plasmid, pADB-PTR55, was sequenced to ensure that the fusion had occurred in frame (see Fig. 1). In vitro transcription-translation revealed that the chimeric gene was translated into a protein of the appropriate size.()


Figure 1: Design of the PDGFR-TR55 receptor chimera. The PDGFR provides the ligand binding and transmembrane domain, while the cytoplasmic domain of TR55 provides the interface to TNF-specific signal transduction components. Nucleotide as well as amino acid sequences of the junction region are indicated. The PDGFR and TR55 fragments were joined via Klenow filled-in HindIII sites (outlined). Two new amino acids (QA) were introduced as a result of the fusion.



Cell Culture and Transfections

NMuMG cells (32) were obtained from ATCC. NMuMG cells stably transfected with the full-length murine PDGF receptor cDNA (cell line RI) were a generous gift from Dr. J. Escobedo (University of California, San Francisco). Cells were grown in DMEM supplemented with 10% bovine calf serum, 50 µg/ml penicillin, and 50 µg/ml streptomycin (Life Technologies, Inc.).

Stably transfected NMuMG cells expressing the PDGFR-TR55 chimeric receptor were obtained by cotransfection of pADB-PTR55 with BMGNeo (33) using electroporation at 960 microfarads/280 volts and subsequent selection with 1000 µg/ml Geneticin (Life Technologies, Inc.). Transfectants expressing vector alone without insert were generated by cotransfecting pAD-CMV1 with BMGNeo.

Radioligand Binding Assays and Scatchard Analyses

Cells were detached by incubation in PBS, 2 mM EDTA at 37 °C for 15 min, washed, and resuspended in cold PBS with 0.2% FCS and 0.02% sodium azide. Assays were set up in triplicate as follows: 1 ng of I-radiolabeled human recombinant BB-chain PDGF (DuPont NEN; specific activity 1480 kBq/µg) or human recombinant TNF- (DuPont NEN; specific activity 1630 kBq/µg) was added to 3 10 cells in a total volume of 300 µl of PBS/FCS/azide with or without a 1000-fold excess of unlabeled ligand. Cells were incubated at 0 °C for 2 h and washed twice in cold PBS/FCS/azide, and total as well as nonspecific binding was analyzed in a -counter (Canberra Packard).

For Scatchard analyses, cells were incubated with serial dilutions of labeled ligand (0.125-5 ng/well) in a total volume of 300 µl of PBS/FCS/azide with or without unlabeled ligand. Cells were washed twice in cold PBS/FCS/azide and analyzed in a -counter (Canberra Packard).

Immunoblots

Cells were lysed in TNE buffer (50 mM Tris, pH 8.0, 1% Nonidet P-40, 2 mM EDTA) containing 10 µg/ml aprotinin/leupeptin. The cell lysates were denatured in sample buffer and boiled, and 25 µg of cell protein per lane were resolved by electrophoresis on 7% SDS-polyacrylamide gels. After electrophoretic transfer to nitrocellulose, reactive proteins were detected using anti-PDGF receptor monoclonal antibodies (Affinity Research Products) and the ECL detection kit (Amersham Corp.).

Proliferation Assays

2 10 cells/well were seeded in 24-well plates and kept in DMEM with 5% FCS for 3 days. After further incubation in quiescing medium (DMEM with 0.1% bovine serum albumin, 5 µg/ml transferrin) for 1 day, cells were stimulated by adding 2 nM human recombinant BB-chain PDGF or 0.6 nM murine recombinant TNF- for 16 h, pulsed with 18.5 kBq [methyl-H]thymidine (DuPont NEN) for 1 h, washed in PBS three times, lysed in 0.1% SDS, and counted in a scintillation counter (Canberra Packard).

Cytotoxicity Assays

10 cells were seeded in flat-bottom 96-well plates and incubated overnight in 100 µl of DMEM, 10% FCS to allow cells to adhere. The cells were then preincubated in medium with 100 ng/ml actinomycin D for 2 h before serial dilutions of murine recombinant TNF- or human recombinant BB-chain PDGF were added. After 18 h, 20 µl of MTT (Sigma, 2.5 mg/ml in PBS) were added, and incubation was continued for an additional 2 h to allow metabolization of MTT to 3-[4,5-dimethylthiazol-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).

Stimulation of Cells, Preparation of Nuclear Extracts, and Electrophoretic Mobility Shift Assays for NF-B

3 10 cells/10-cm dish were stimulated by adding human recombinant BB-chain PDGF or murine recombinant TNF- to a final concentration of 10 nM (PDGF) or 0.6 nM (TNF). The cells were incubated at 37 °C for 20 min or for the indicated time, washed twice in ice-cold PBS, and harvested with a cell scraper.

Nuclear extracts were prepared as described(34) . Electrophoretic mobility shift assays were performed by incubating 3 µg of nuclear extract with 4 µg of poly(dI-dC) (Pharmacia Biotech Inc.) in binding buffer (5 mM HEPES, pH 7.8, 5 mM MgCl, 50 mM KCl, 0.5 mM dithiothreitol, 10% glycerol in a total volume of 20 µl for 20 min at room temperature. Then, end-labeled double-stranded oligonucleotide probe (NF-B-specific oligonucleotide containing two tandemly arranged NF-B binding sites of the HIV-1 long terminal repeat enhancer (5`-ATCAGGGACTTTCCGCTGGGGACTTTCCG-3`), 1 10 to 5 10 cpm) was added, and the reaction mixture was incubated for 7 min. The samples were separated on native 6% polyacrylamide gels in low ionic strength buffer (0.25 Tris borate-EDTA). For competition experiments, unlabeled or a mutated oligonucleotide containing altered B sites (5`-GATCACTCACTTTCCGCTTGCTCACTTTCCAG-3`) was added before the initial 20-min incubation.


RESULTS

The PDGFR-TR55 receptor was generated by fusing the extracellular and transmembrane domain of the murine PDGF receptor to amino acids 243-426 of the human p55 TNF receptor cytoplasmic domain (see Fig. 1). The chimeric receptor was cloned into the expression vector pAD-CMV1 under the transcriptional control of the cytomegalovirus promoter and 5` to the simian virus 40 polyadenylation site. The resulting plasmid was designated pADB-PTR55 (see ``Materials and Methods'').

A transient expression system was initially utilized to monitor induction of NF-B in NMuMG cells transfected with pADB-PTR55 and a 4 immunoglobulin/HIV-B chloramphenicol acetyltransferase reporter plasmid(35) . Due to low transfection efficiencies, however, this system was not suitable to conduct detailed studies of the potential signal transduction through the PDGFR-TR55 chimera (data not shown).

Therefore, parental NMuMG cells, which neither express detectable PDGF receptor mRNA nor PDGF receptors at the cell surface, were stably transfected with plasmid pADB-PTR55, and Geneticin-resistant colonies were cloned. A total of 32 clones was analyzed for cell surface expression of the PDGFR-TR55 chimeric receptor in binding assays for I-PDGF. Three positive clones (designated PT55-1 to PT55-3) were characterized further. As a control, transfectants were selected that contain the expression vector pAD-CMV1 without insert. Of those, clone PAD-1, together with parental NMuMG cells, served as a negative control in the subsequent experiments.

Parental NMuMG cells, clones PAD-1, PT55-1 to PT55-3, and the NMuMG transfectant line RI expressing the full-length PDGF receptor were analyzed for expression of endogenous murine p55 TNF receptors using human I-TNF. All cell lines examined specifically bound I-TNF, confirming endogenous TR55 expression (Fig. 2A). When analyzed for the binding of I-PDGF, both NMuMG and PAD-1 cells proved negative, while RI cells demonstrated significant binding. Clones PT55-1 to PT55-3 did also prove positive for binding of I-PDGF, though at a lower level than that seen with the full-length PDGF receptor in RI cells (Fig. 2B).


Figure 2: Expression of TR55, PDGFR, and PDGFR-TR55 by parental and transfected NMuMG cells. A, binding of I-labeled TNF- to endogenous p55 TNF receptors of parental NMuMG cells, cells transfected with pAD-CMV1 without insert (PAD-1), cells expressing the full-length PDGF receptor (RI), or transfectants expressing the PDGFR-TR55 chimeric receptor (PT55-1 to PT55-3). B, binding of I-labeled PDGF-BB to the full-length PDGF receptor or the PDGFR-TR55 receptor chimera. The filledcolumns indicate total binding (TB), and the opencolumns indicate nonspecific binding in the presence of excess unlabeled ligand (NSB). Errorbars represent the standard deviations from three experiments. C, Scatchard plot of specific I-PDGF binding to the PDGFR-TR55 receptor chimera on PT55 cells or to the full-length PDGF receptor on RI cells. The lines were plotted by linear regression analysis (PT55-1: r = -0.96; PT55-2: r = -0.94; PT55-3: r = -0.98; RI: r = -0.93). Each value represents the mean of triplicate experiments. Receptor numbers per cell and the K value for each cell line are indicated. D, Western blot analysis of the PDGFR-TR55 chimeric receptor in NMuMG transfectants. Protein extracts from parental NMuMG cells, PAD-1 cells, RI cells, and clones PT55-1 to PT55-3 were separated on 7% SDS-polyacrylamide gels and immunoblotted using a monoclonal PDGFR antibody (Affinity Research Products). The arrow indicates the predicted size for the receptor chimera.



Scatchard analyses of clones PT55-1, PT55-2, and PT55-3 demonstrated high affinity binding of radiolabeled PDGF-BB to the PDGFR-TR55 receptor (Fig. 2C), comparable to the affinity described for PDGF binding to the wild type PDGF receptor on murine 3T3 cells(36) . In addition, Scatchard analyses of RI cells demonstrated an almost identical binding affinity for the transfected wild type PDGF receptor (Fig. 2C), indicating that the reduced levels of I-PDGF binding observed with PT55 cells result from reduced numbers of the PDGFR-TR55 molecule rather than from an impaired binding capability of the receptor chimera. Reduced expression of PDGFR-TR55 is probably due to toxic effects of a highly expressed TR55 cytoplasmic domain(37) .

The expression of the PDGFR-TR55 chimeric receptor by clones PT55-1 to PT55-3 was additionally confirmed by fluorescence-activated cell sorting analysis using antibody SM14 directed against the extracellular domain of the PDGF receptor (data not shown).

Western blots using a monoclonal PDGF receptor antibody demonstrated the presence of a reactive band of 78 kDa, the predicted size for the PDGFR-TR55 chimera (Fig. 2D). As expected, this 78-kDa protein was not detected in NMuMG, RI, or PAD-1 cells. The full-length PDGF receptor in RI cells was masked by the presence of a cross-reactive band comigrating with the PDGF receptor. This band was observed in parental NMuMG cells as well as in 70Z/3 pre-B-cells, which both are negative for PDGF receptor expression (data not shown).

PDGF is a potent mitogen for many cell types, while TNF exerts both proliferative and cytotoxic effects depending on the target cell. To determine the type of response the PDGFR-TR55 chimeric receptor would convey, we analyzed proliferative and cytotoxic effects of PDGF and TNF on parental NMuMG cells, PAD-1, RI, and PT55 transfectants.

First, cells were assayed for PDGF- or TNF-mediated induction of DNA synthesis by [H]thymidine incorporation assays (Fig. 3). Engagement of the endogenous TR55 by TNF did not induce any significant proliferation of either cell line. PDGF induced DNA synthesis in RI cells expressing the full-length PDGF receptor. In contrast, PDGF did not stimulate the proliferation of parental NMuMG cells, PAD-1 cells, or of clones PT55-1 to PT55-3 expressing the PDGFR-TR55 chimera. These results indicate that neither endogenous TR55 nor the PDGFR-TR55 receptor are able to mediate a proliferative response in these cells.


Figure 3: Proliferative effects of TNF and PDGF on NMuMG transfectants. Parental NMuMG cells, PAD-1 cells, RI cells expressing the full-length PDGF receptor, and PT55 cells expressing the PDGFR-TR55 chimera were kept in serum-free medium or stimulated with TNF or PDGF or serum. [H]Thymidine incorporation into cellular DNA is shown relative to serum-stimulated cells (100%). Errorbars indicate the standard deviations derived from triplicate determinations. The actual cpm values obtained in these experiments for serum-stimulated cells were: NMuMG cells, 40618 ± 2773; PAD-1 cells, 16801 ± 3304; RI cells, 21749 ± 3055; PT55-1 cells, 27444 ± 625; PT55-2 cells, 9059 ± 1972; PT55-3 cells, 10003 ± 2066. The data shown are representative of a total of three independent experiments.



TNF treatment of parental and transfected NMuMG cells resulted in cytotoxic effects (Fig. 4A), which is likely mediated through endogenous TR55. When PT55 clones expressing the PDGFR-TR55 chimera were treated with PDGF, a pronounced cytotoxic effect was elicited. In contrast, parental NMuMG, PAD-1, or RI cells did not show a cytotoxic response to PDGF (Fig. 4B).


Figure 4: Cytotoxicity mediated through TR55 or PDGFR-TR55. A, parental NMuMG cells, RI cells, PAD-1 cells (leftpanel), and PT55 clones 1-3 (rightpanel) were treated with serial dilutions of TNF. B, parallel analysis using serial dilutions of PDGF. Errorbars indicate the standard deviations obtained from six parallel experiments.



We finally examined whether the PDGFR-TR55 chimera was capable to mediate the activation of the transcription factor NF-B, an important mediator of TNF responses.

As shown in Fig. 5(lanes1-6), nuclear NF-B activity in parental NMuMG and PAD-1 cells was markedly increased after stimulation of cells with TNF but not after PDGF treatment. In PT55 cells, NF-B activation could be induced by both TNF and PDGF (Fig. 5, lanes7-24), and the kinetics of NF-B activation were similar in any instance (data not shown). Competition experiments using oligonucleotides containing wild type or mutated NF-B sites confirmed NF-B binding activity induced by PDGF (Fig. 5, lanes17 and 18).


Figure 5: PDGF induces NF-B in PT55 cells. Parental NMuMG cells and PAD-1 cells were left unstimulated (lanes1 and 4) or treated with either TNF (lanes2 and 5) or PDGF (lanes3 and 6) for 20 min as described under ``Materials and Methods.'' PT55-1 cells were left untreated (lane7) or stimulated with TNF for 20 min (lane8, positive control). Alternatively, PT55-1 cells were stimulated with PDGF for the indicated times (lanes9-16) or for 20 min with addition of wild type (lane17) or mutated (lane18) NF-B oligonucleotides for competition. Lanes19-24, same experiment as in lanes1-6, using cell lines PT55-2 and PT55-3.




DISCUSSION

The results of our study indicate that two key responses of TNF signaling pathways, cytotoxicity and induction of NF-B, can be mediated solely by the TR55 cytoplasmic domain without requirement for extracellular or transmembrane sequences. Furthermore, dimerization of the TR55 cytoplasmic domain by a bivalent ligand, PDGF, is sufficient to activate the intrinsic functions of the receptor. This mode of activation ranges TR55 among the group of other receptors that are dimerized prior to activation, e.g. the epidermal growth factor receptor, the PDGFR, and many more(21) .

Controversial data exist on the conformation of biologically active TR55. Earlier studies reported that TNF acts as a trimer (38) that may induce the formation of both TR55 dimers and trimers. It is widely acknowledged from x-ray diffraction studies (23, 24, 25) that TR55 can form trimers after ligand binding. However, these studies have been performed on TR55 crystals and do not rule out the possibility that TR55 adopts other conformations (e.g. dimers) at the cell surface when bound to ligand. Active signaling of TR55 dimers might be deduced from cross-linking experiments with agonistic antibodies(39, 40) . However, antibody cross-linking does not exclude the formation of trimeric or multimeric complexes. Obviously, antibody-mediated receptor clustering does not always correctly reflect results obtained by using physiologic ligands, which is illustrated by experiments in which the cytoplasmic domains of TR55 or Fas were fused to the extracellular domain of CD40. Cross-linking with agonistic CD40 antibodies did not activate the CD40-TR55 fusion proteins(30) .

Indeed, not any physiologic ligand can activate the TR55 cytoplasmic domain. A hybrid receptor consisting of the TR55 cytoplasmic domain fused to extracellular sequences of the CD44 hyaluronate receptor was incapable of inducing cell death after ligand-mediated cross-linking, supporting the conclusion that TR55 requires a specific type of oligomerization different from that of CD44(30) .

While oligomerization through CD44 is apparently unable to activate TR55, PDGF-mediated cross-linking appears perfectly sufficient to activate the TR55 cytoplasmic domain. PDGF represents a classical dimeric ligand, that is, the plethora of data indicates that PDGF activates its receptor by dimerizing two receptor molecules(28, 29) . The same holds apparently true for the PDGFR-TR55 chimeric receptor.

Since TR55 dimerization is obviously sufficient to elicit cytotoxicity and induction of NF-B, one might speculate that signaling through a dimeric p55 TNF receptor employs the same pathways as signaling through the TR55 trimer. More knowledge is needed about the signaling events that take place following TR55 dimerization, e.g. which proteins associate with TR55, and if those proteins represent the same set that binds to an activated TR55 trimer or if different sets of associated proteins exist for different states of TR55 clustering. The identification and cloning of those proteins will provide essential information about the p55 TNF receptor signal transduction pathways.


FOOTNOTES

*
This work was supported by grants from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Institut für Immunologie, Christian-Albrechts-Universität Kiel, Brunswiker Str. 4, 24105 Kiel, Germany.

To whom correspondence should be addressed. Tel.: 49-431-597-3339; Fax: 49-431-597-3335.

The abbreviations used are: TNF, tumor necrosis factor; TR, tumor necrosis factor receptor; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; FCS, fetal calf serum; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.

D. Adam, U. Keler, and M. Krönke, unpublished observation.


ACKNOWLEDGEMENTS

We thank H. Wagner for continuous support.


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