Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Osteoprotegerin Is a Receptor for the Cytotoxic Ligand TRAIL

Osteoprotegerin Is a Receptor for the Cytotoxic Ligand TRAIL THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 23, Issue of June 5, pp. 14363–14367, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. (Received for publication, January 26, 1998 and in revised form, March 9, 1998) John G. Emery‡, Peter McDonnell‡, Michael Brigham Burke¶, Keith C. Deeni, Sally Lyn‡, Carol Silverman**, Edward Dul‡‡, Edward R. Appelbaum‡‡, Chris Eichmani, Rocco DiPrinzioi, Robert A. Dodds§§, Ian E. James§§, Martin Rosenberg¶¶, John C. Lee§§, and Peter R. Young‡§ From the Departments of ‡Molecular Biology, ¶Structural Biology, iMolecular and Cellular Immunology, **Protein Biochemistry, ‡‡Gene Expression Sciences, and §§Bone and Cartilage Biology at SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406 and the ¶¶Department of Anti-infectives at SmithKline Beecham Pharmaceuticals, Collegeville, Pennsylvania 19426 TRAIL is a tumor necrosis factor-related ligand that member that increases bone density and causes splenomegaly when overexpressed in transgenic mice (14). OPG inhibits os- induces apoptosis upon binding to its death domain- containing receptors, DR4 and DR5. Two additional teoclastogenesis in vitro, suggesting that increased bone den- TRAIL receptors, TRID/DcR1 and DcR2, lack func- sity results from decreased numbers of mature osteoclasts. It tional death domains and function as decoy receptors has been suggested that OPG may neutralize a TNF-related for TRAIL. We have identified a fifth TRAIL receptor, ligand that promotes osteoclast differentiation or bind a mem- namely osteoprotegerin (OPG), a secreted tumor ne- brane-anchored TNF-related ligand that regulates osteoclasto- crosis factor receptor homologue that inhibits osteo- genesis via reverse signaling (14). clastogenesis and increases bone density in vivo. To identify interactions between novel TNF ligand and re- M, which OPG-Fc binds TRAIL with an affinity of 3.0 n ceptor family members, we screened a panel of receptor-Fc is slightly weaker than the interaction of TRID-Fc or fusion proteins for binding to TRAIL and have identified a DR5-Fc with TRAIL. OPG inhibits TRAIL-induced apo- novel interaction between OPG and TRAIL. We demonstrate ptosis of Jurkat cells. Conversely, TRAIL blocks the that OPG binds to TRAIL in vitro and can block TRAIL- anti-osteoclastogenic activity of OPG. These data sug- induced apoptosis. Conversely, TRAIL appears to block OPG- gest potential cross-regulatory mechanisms by OPG mediated inhibition of osteoclastogenesis in vitro. These results and TRAIL. suggest that OPG and TRAIL may function to inhibit each other. Members of the TNF ligand and receptor superfamilies play EXPERIMENTAL PROCEDURES an important role in regulating many biological processes, in- Preparation of Proteins and Antibodies—All cDNAs were identified cluding cytokine production, apoptosis, cell activation, lympho- by homology searches of an assembled Expressed Sequence Tag data cyte co-stimulation, proliferation, immunoglobulin secretion, base, and the 59-most cDNA clones were obtained. The cDNAs encoding and isotype switching (1). Three of the ligands, FasL, TNFa, the native leader peptides and extracellular domains of OPG (amino acids 1– 401) (14), herpes virus entry mediator (HVEM) (amino acids and TRAIL/Apo-2L, which are type II transmembrane proteins, 1–192) (15–17), DR3 (amino acids 1–199) (11, 18 –20), DR5 (amino acids exhibit potent cytotoxic activity, inducing apoptosis of suscep- 1–133 of Ref. 7) (7–12), and TRID (amino acids 1–240) (7–10) were tible cells within hours (2– 4). These cytotoxic ligands bind to polymerase chain reaction-amplified and subcloned upstream of an and aggregate type I transmembrane receptors with cytoplas- in-frame Factor Xa protease cleavage site and the hinge-Fc region of a mic “death domains,” which ultimately activate a protease cas- human IgG-g1 heavy chain in COSFclink (21, 22). The IL5R-Fc con- cade leading to apoptosis (5). struct was described previously (22). OPG-Fc, HVEM-Fc, DR3-Fc, and TRID-Fc were purified from conditioned medium of Chinese hamster Recently four receptors for TRAIL have been identified. Two ovary stable transfectants by protein G affinity chromatography (Am- of these receptors, DR4 (6) and DR5/TRICK2/TRAIL-R2 (7–12), ersham Pharmacia Biotech). Samples contaminated with Fc dimer were contain cytoplasmic death domains and are capable of inducing further purified on Superdex 200 (Amersham). DR5-Fc protein was apoptosis upon overexpression in transfected cells. Addition- purified from the conditioned medium of COS cell transient transfec- ally, two decoy receptors, DcR1/TRID/TRAIL-R3 (7–10) and tants by affinity chromatography on Prosep A (Bioprocessing Ltd.). DcR2 (13), lack functional death domains and inhibit TRAIL- Soluble receptors without the Fc domain were prepared by digesting with 1/80 e/s Factor Xa (Hematologic Technologies, Inc.) at 4 °C for 72 h. induced apoptosis. The existence of decoy receptors for TRAIL Fc dimer was removed by protein A affinity chromatography helps explain the absence of massive apoptosis in cells and (Amersham). tissues that express both TRAIL and one of its cytotoxic recep- Soluble TRAIL tagged at the N terminus with the FLAG epitope tors (7). However, in tissues lacking the decoy receptor, another (TRAIL-Flag, amino acids 95–281) (4) was constructed in the pCDN mechanism must exist to prevent TRAIL-induced apoptosis. mammalian expression vector (23) that had been modified to contain an Osteoprotegerin (OPG) is a secreted TNF receptor family in-frame tissue plasminogen activator signal sequence upstream of the FLAG-TRAIL coding region. Soluble TRAIL-Flag was purified from 30 liters of conditioned medium from a Chinese hamster ovary stable * The costs of publication of this article were defrayed in part by the transfectant by sequential chromatography on hydroxyapatite (Bio- payment of page charges. This article must therefore be hereby marked Rad, type I Macro Prep), Q-Sepharose, Mono Q, and Superdex 200 “advertisement” in accordance with 18 U.S.C. Section 1734 solely to (Amersham). TRAIL-Flag eluted at ;90 kDa, slightly larger than ex- indicate this fact. pected for a trimer, but appeared to be predominantly trimeric by § To whom correspondence should be addressed: Dept. of Molecular analytical ultracentrifugation analysis. Biology, Mail Code UW2101, SmithKline Beecham Pharmaceuticals, Polyclonal antibodies to TRAIL were raised in rabbits by injection of 709 Swedeland Rd., King of Prussia, PA 19406. Tel.: 610-270-7691; Fax: TRAIL (amino acids 41–281) that contained an epitope tag and hexa- 610-270-5114; E-mail: [email protected]. The abbreviations used are: TNF, tumor necrosis factor; OPG, os- teoprotegerin; ELISA, enzyme-linked immunosorbent assay; TRAP, tartrate-resistant acid phosphatase. P. Hensley, unpublished data. This paper is available on line at http://www.jbc.org 14363 This is an Open Access article under the CC BY license. 14364 Osteoprotegerin Is a Receptor for TRAIL histidine tag at its N terminus. The tagged TRAIL protein was ex- unlabeled TRAIL-Flag. Complexes were captured with 30 ml of protein pressed in Escherichia coli as insoluble inclusion bodies using the pAS A-Sepharose 4B, washed twice in binding buffer, and electrophoresed expression system (24), solubilized in guanidine HCl, and purified by through 10% SDS-polyacrylamide gel electrophoresis. Gels were fixed nickel nitrilotriacetic acid chromatography. for 30 min, incubated with Enlighten (Amersham) for 30 min, and dried Receptor Precipitations—TRAIL-Flag (250 ng) was added to 2 mgof before autoradiography. receptor-Fc in binding buffer (25 mM HEPES, pH 7.2, 0.25% bovine ELISAs—ELISAs were performed according to standard techniques serum albumin, 0.01% Tween in RPMI 1640) and incubated for2hon (25). Immunlon 4 (Dynatech Laboratories) plates were coated overnight ice. Protein A-Sepharose 4B (Amersham, 30 ml of a 75% slurry) was at 4 °C with anti-Flag M2 monoclonal antibody (Eastman Kodak Co.) (2 added and incubated for1hon ice. Complexes were recovered by mg/ml, 100 ml/well) in 0.05 M bicarbonate buffer, pH 8.6. After washing centrifugation, washed three times with binding buffer, electrophoresed and blocking with 0.5% gelatin in phosphate-buffered saline, TRAIL- on 15% SDS-polyacrylamide gel electrophoresis, and transferred to Flag (2 mg/ml, 100 ml/well) was bound for1hat room temperature. nitrocellulose for Western analysis with an anti-TRAIL polyclonal an- Serial dilutions of OPG-Fc or DR5-Fc (1 mg/ml to 5 ng/ml, 100 ml/well) tiserum. For binding reactions with full-length TRAIL, the entire were added after washing and incubated for1hat room temperature. TRAIL coding region was polymerase chain reaction-amplified and The Fc fusions were detected with a biotinylated goat anti-human IgG cloned into pCDN (23). HEK293 cells (3 3 10 ) were transfected with 10 antibody (Southern Biotechnology Associates) (1/2000 dilution, 100 ml/ mg of pCDN-TRAIL or pCDN alone using lipofectAMINE reagent (Life well), horseradish peroxidase-conjugated streptavidin (Southern Bio- Technologies, Inc.). 48 h after transfection, regular growth medium was technology Associates) (1/4000 dilution, 100 ml/well), and ABTS perox- replaced with methionine/cysteine-free Dulbecco’s modified Eagle’s me- idase substrate (100 ml/well, Kirkegaard and Perry Laboratories). The dium. After a 1-h starvation, 1.5 mCi of translabel [ S]Met,Cys (ICN) was added to transfected cells along with 2% dialyzed fetal bovine TABLE I serum for 6 h. Cells were washed in Dulbecco’s modified Eagle’s me- Binding constants for the interaction of TRAIL-Flag with OPG-Fc, dium and lysed in 1 ml of STET (150 mM NaCl, 10 mM Tris, pH 7.5, 1 TRID-Fc, and DR5-Fc mM EDTA, 1% Triton X-100, 10 mg/ml leupeptin, 20 mg/ml aprotinin, 2 Receptor k k Calculated K a d D mM phenylmethylsulfonyl fluoride) for 20 min on ice. Lysates were 21 21 21 centrifuged, and the supernatants were precleared with 100 ml of pro- M s s nM tein A-Sepharose 4B (Amersham). 50 ml of the precleared lysate was 4 24 OPG-Fc 6.6 3 10 2.0 3 10 3.0 used in receptor precipitation with 2 mg of OPG-Fc as described above. 5 24 TRID-Fc 2.9 3 10 3.2 3 10 1.1 Binding volumes were increased to 1 ml, and the binding reaction 5 24 DR5-Fc 3.4 3 10 2.6 3 10 0.76 proceeded overnight at 4 °C. Competition was achieved with 2 mgof FIG.1. OPG binds to TRAIL. A, immunoprecipitation of TRAIL-Flag by receptor-Fc proteins. TRAIL-Flag was incubated with the indicated receptor-Fc proteins, and complexes were precipitated with protein A-Sepharose. The samples were analyzed by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose for Western analysis with an anti-TRAIL polyclonal antiserum. B, cleaved OPG, TRID, and DR5 compete with Fc fusions for binding to TRAIL. Binding reactions were essentially as described above, except that a 10-fold molar excess of cleaved receptor was added immediately before the Fc fusion. C, OPG-Fc binds to cell-associated full-length TRAIL (FL-TRAIL). 293 cells were transiently transfected with a full-length TRAIL expression construct and labeled with [ S]methionine, and cell lysates were subject to immunoprecipitation. Unlabeled soluble TRAIL-Flag was added as above where indicated. D, OPG- and DR5-Fc bind to TRAIL-Flag in ELISA. Microtiter plates were coated with an anti-Flag M2 monoclonal antibody and allowed to bind TRAIL-Flag. The plates were incubated with serial dilutions of OPG-Fc (M) and DR5-Fc (l ) proteins, and binding was detected with a biotinylated goat anti-human IgG antibody followed by streptavidin-horseradish peroxidase and ABTS peroxidase substrate. E, OPG and DR5 compete with OPG-Fc for binding to TRAIL-Flag. The assay was performed essentially as described above, except that serial dilutions of cleaved competitor OPG (M) or DR5 (l ) were incubated with the anti-Flag-captured TRAIL-Flag before addition of OPG-Fc. Samples without competitor are indicated by ‚. s, soluble; KD, kilodaltons. Osteoprotegerin Is a Receptor for TRAIL 14365 FIG.2. OPG blocks TRAIL-induced apoptosis. Jurkat cells were treated with increasing amounts of OPG (A), DR5-Fc (B), or DR3-Fc (C) in the presence of TRAIL-Flag plus anti-FLAG M2 mono- clonal antibody (Kodak) or the anti-Fas agonist monoclonal antibody CH-11 (MBL) for 3 h. Cytotoxicity was assessed by de- termining the percentage of propidium iodide (PI)-permeable cells (mean 6 S.D., n 5 2) by fluorescence-activated cell sorter analysis. D and E, cytospins of Jurkat cells treated with TRAIL-Flag as described above in the absence (D)or presence (E) of OPG (5 mg/ml) were stained with 49,6-diamidine-29-phenylin- dole dihydrochloride, and the nuclei were examined by fluorescence microscopy. Note the dramatic reduction in nuclear fragmentation in OPG-treated cells. plates were read at 405 nm on a UV max microplate reader (Molecular 7 days, after which the media were removed, and the cell monolayer Devices). For competition assays, serial dilutions of OPG or DR5 was stained for tartrate-resistant acid phosphatase (TRAP) or by a without Fc domains (50 mg/ml to 30 ng/ml, 100 ml/well) were added to TRAP-solution assay (29). In the TRAP-solution assay, enzyme activity the plate for1hat room temperature before the addition of OPG-Fc (10 was measured by the conversion of p-nitrophenylphosphate (20 nM)to mg/ml, 10 ml/well). p-nitrophenol in the presence of 80 mM sodium tartrate and expressed Determination of the Affinity of TRAIL for Its Receptors—The as- as OD405 units. sociation and dissociation rates of the interaction of TRAIL with RESULTS AND DISCUSSION captured receptor fusion proteins were determined by surface plas- mon resonance using a BIAcore 1000 (BIAcore Inc.). The capture To identify interactions between novel TNF ligand and re- surface was a protein A (Pierce) modified CM5 sensor chip (26). The ceptor family members, we screened a panel of receptor-Fc sensor surface was equilibrated with a buffer of 20 mM sodium phos- fusion proteins for binding to TRAIL by immunoprecipitation. phate, 150 mM sodium chloride, and 0.005% Tween 20, pH 7.4, and TRAIL bound to TRID-Fc and DR5-Fc proteins as expected analyses were performed at 30 ml/min at 25 °C. The receptor-Fc (Fig. 1A). TRAIL also bound to OPG-Fc. TRAIL did not bind to fusion protein was diluted into the above buffer to 1 mg/ml, and a 20 ml injection was passed over the capture surface, followed by a 150-ml DR3-Fc and HVEM-Fc (Fig. 1A), two additional members of the injection of the TRAIL-Flag. After the association phase, 500 s of TNF receptor superfamily, indicating that the OPG-TRAIL dissociation data was collected. The surface was regenerated after interaction is not Fc-mediated. To confirm that binding of each cycle. Sets of 3– 4 analyte concentrations, 500 20 nM, were OPG-Fc to TRAIL was not to the Fc region, a 10-fold molar collected and analyzed by nonlinear regression analysis (27) using the excess of OPG cleaved of its Fc domain was incubated with BIAevaluation software 2.1. The dissociation data was fitted on the TRAIL and the Fc fusion proteins before immunoprecipitation. basis of the AB 5 A 1 B model. The association data was fitted to A 1 B 5 AB using the type 1 model. Cleaved OPG, TRID, and DR5 effectively competed away the Apoptosis Assays—Jurkat cells (ATCC) (5 3 10 cells/ml) were binding of OPG-Fc to TRAIL (Fig. 1B). To determine if OPG treated with increasing concentrations of OPG (0, 0.2, 1.0, or 5.0 mg/ml), could bind to cell-associated TRAIL, HEK293 cells were tran- DR5-Fc (0, 0.008, 0.04, and 0.2 mg/ml), or DR3-Fc (0, 0.2, 1.0, or 5.0 siently transfected with a full-length TRAIL expression vector, mg/ml), and apoptosis was induced with an anti-Fas CH-11 monoclonal labeled with [ S]methionine, and the cell lysate was subjected antibody (0.1 mg/ml, Medical and Biological Laboratories) or TRAIL- to immunoprecipitation by the OPG-Fc fusion protein. OPG-Fc Flag (0.1 mg/ml) with anti-FLAG M2 monoclonal antibody (3 mg/ml, Kodak) for 3 h. The anti-Flag M2 antibody was added to all treatments precipitated a protein of the expected size in TRAIL but not to normalize for potential nonspecific effects. Cells were stained with vector-transfected cells (Fig. 1C), indicating that OPG-Fc binds propidium iodide (6.5 mg/ml, Boehringer Mannheim) and quantitated to cell-associated TRAIL. Unlabeled soluble TRAIL-Flag effec- by fluorescence-activated cell sorter analysis. Cytospins of Jurkat cells tively competed with the S-labeled full-length TRAIL for were fixed for 30 min in ice-cold 1% paraformaldehyde in phosphate- binding to OPG-Fc. buffered saline, washed in phosphate-buffered saline, and stained for The ability of TRAIL to bind OPG was also examined by 30 min with 49,6-diamidine-29-phenylindole dihydrochloride (1 mg/ml, Boehringer Mannheim). ELISA. Both OPG-Fc and DR5-Fc proteins were capable of Osteoclastogenesis Assays—A modified osteoclast formation assay binding to anti-Flag-immobilized TRAIL-Flag (Fig. 1D). In was adapted from Ref. 28. In brief, bone marrow mononuclear cells were blocking assays, cleaved DR5 prevented OPG-Fc from binding isolated from femurs of 4 – 6-week DBF1 mice in the presence of early to TRAIL-Flag (Fig. 1E), indicating that the DR5 and OPG passage stromal cells obtained from a human osteoclastoma at a 400:1 binding sites overlap. Cleaved OPG was more effective than ratio. The co-culture was established in RPMI 1640 supplemented with cleaved DR5 in blocking TRAIL-Flag binding (Fig. 1E), possibly 10% fetal calf serum, 100 nM dexamethasone, 10 nM vitamin D3. Test reagents were added at day 0, and the cultures were left undisturbed for reflecting the fact that the cleaved OPG is a dimer, whereas 14366 Osteoprotegerin Is a Receptor for TRAIL Anti-Flag-aggregated TRAIL-Flag exhibited significant cyto- toxicity within 3 h (Fig. 2A). OPG demonstrated a dose-depend- ent inhibition of TRAIL-induced cytotoxicity and blocked the appearance of apoptotic nuclei in Jurkat cells treated with TRAIL-Flag (Fig. 2, D and E). However, OPG did not prevent killing by an agonist anti-Fas monoclonal antibody, another potent inducer of apoptosis, indicating that OPG does not ac- tivate a nonspecific survival pathway (Fig. 2A). DR5-Fc exhib- ited a similar but more potent inhibition of TRAIL-induced cytotoxicity (Fig. 2B), likely due to its higher affinity for TRAIL. DR3-Fc, which does not bind TRAIL, did not inhibit TRAIL-induced killing (Fig. 2C). This data suggests that OPG, which is secreted and detectable in the circulation (14), may be a soluble antagonist receptor for TRAIL. OPG may negatively regulate TRAIL activity, similar to the inhibition of TNFa by secreted TNF receptors of poxviruses (35). Furthermore, TNF receptors are shed from the cell surface and found in the circulation following certain stimuli. It has been suggested that soluble TNF receptors may inhibit or clear circulating TNFa (36). A similar role for OPG might also underlie the splenomeg- aly in mice that overexpress OPG (14). In mice lacking the anti-apoptotic Bcl-2 gene, there is a decrease in the size of the spleen, suggesting that spleen cell homeostasis is regulated by apoptotic mechanisms (37). Thus, if TRAIL is involved in reg- ulation of spleen cell homeostasis by inducing apoptosis, then constitutive overexpression of OPG might result in decreased apoptosis and splenomegaly. OPG inhibits osteoclastogenesis in vitro (14). To determine the significance of the OPG-TRAIL interaction in osteoclasto- genesis, we examined the effects of TRAIL in an in vitro osteo- clastogenesis assay. Human stromal cells were co-cultured with murine bone marrow cells for 7 days in the presence of OPG, TRAIL, or TL4, another TNF-like ligand that does not bind OPG. OPG inhibited the formation of multinucleate TRAP positive osteoclasts as expected, with an IC of approx- imately 60 pM (Fig. 3). TRAIL alone had no effect on osteoclas- togenesis. However, TRAIL completely blocked the inhibitory effect of OPG (Fig. 3), suggesting that soluble TRAIL may function to regulate the activity of OPG. TL4 had no effect on osteoclastogenesis in the presence or absence of OPG. It has been suggested that OPG may inhibit osteoclastogen- esis by binding to a pro-osteoclastogenic TNF-related ligand (14). Our data suggest that this ligand may not be TRAIL. First, soluble TRAIL alone had no effect on osteoclastogenesis, FIG.3. TRAIL blocks the ability of OPG to inhibit osteoclasto- and the IC of OPG is approximately 50-fold lower than the genesis. In vitro osteoclastogenesis assays in the absence (A, C, and E) affinity of OPG for soluble TRAIL. Secondly, anti-Flag-aggre- or presence of OPG (10 ng/ml) (B, D,, and F), TRAIL-Flag (C and D), or gated TRAIL, which may mimic membrane TRAIL, induced TL4 (E and F), a novel TNF homologue that does not bind OPG. Arrows massive apoptosis in these co-cultures, which is the opposite in A indicate the multinucleate TRAP-positive osteoclasts. G, quanti- activity to what one would expect for the OPG ligand (data not tation of TRAP levels in osteoclastogenesis assay. shown). However, further experiments will be necessary to cleaved DR5 is a monomer (Ref. 14 and data not shown). fully define the role of TRAIL in osteoclastogenesis and the Although overlapping, the binding sites of OPG-Fc and DR5-Fc existence of other OPG ligands. are not identical, since only OPG-Fc recognized directly immo- The identification of a fifth receptor for TRAIL suggests that bilized TRAIL-Flag (i.e. without an anti-Flag antibody, data complex regulatory mechanisms control its activity. A soluble not shown). antagonist receptor, OPG, and two decoy receptors, DcR1/ The affinity of each receptor for TRAIL was determined by TRID/TRAIL-R3 and DcR2, may prevent the transduction of measuring the kinetics of TRAIL binding to each by surface apoptotic signaling through the death receptors DR4 and DR5/ plasmon resonance (Table I). OPG-Fc binds to TRAIL-Flag TRICK2/TRAIL-R2. It has also been suggested that the gener- with an affinity of 3.0 nM, which is slightly weaker than the ation of two DR5/TRICK2/TRAIL-R2 isoforms by alternate affinities of TRID-Fc and DR5-Fc for TRAIL-Flag (1.1 nM and splicing may regulate cellular responses to TRAIL (11). Addi- 0.76 nM, respectively). The affinities of each receptor are within the range of affinities reported for other physiologically rele- S. Kumar, S. Holmes, J. Fox, S. Gluck, T. Chadderton, and K. B. vant TNF-like ligand and receptor pairs (30 –34). Tan, unpublished data. The observation that OPG binds TRAIL in vitro suggests J. A. Harrop, P. C. McDonnell, M. Brigham-Burke, S. D. Lyn, J. that OPG may inhibit the cytotoxic activity of TRAIL. To ex- Minton, K. B. Tan, K. Dede, J. Spampanato, C. Silverman, P. Hensley, amine this possibility, Jurkat cells were treated with anti-Flag- R. DiPrinzio, J. G. Emery, C. Eichman, M. Chabot-Fletcher, A. Truneh, aggregated TRAIL-Flag in the presence or absence of OPG. and P. R. Young, submitted for publication. Osteoprotegerin Is a Receptor for TRAIL 14367 Davy, E., Bucay, N., Renshaw-Gegg, L., Hughes, T. M., Hill, D., Pattison, tionally, secreted TRAIL itself may antagonize OPG activity or W., Campbell, P., Sander, S., Van, G., Tarpley, J., Derby, P., Lee, R., prevent signaling by membrane-anchored TRAIL through its Program, A. E., and Boyle, W. J. (1997) Cell 89, 309 –319 death receptors, as has been described for FasL and TNFa 15. Montgomery, R. I., Warner, M. S., Lum, B. J., and Spear, P. G. (1996) Cell 87, 427– 436 (reviewed in Ref. 3). A complete understanding of these regu- 16. Hsu, H., Solovyev, I., Colombero, A., Elliott, R., Kelley, M., and Boyle, W. J. latory circuits awaits the determination of soluble TRAIL and (1997) J. Biol. Chem. 272, 13471–13474 OPG levels in vivo and an analysis of knockout mutants of 17. Kwon, B. S., Tan, K. B., Ni, J., Kim, K. K., Kim, Y.-J., Wang, S., Gentz, R., Yu, G.-L., Harrop, J., Lyn, S. D., Silverman, C., Porter, T. G., Truneh, A., and TRAIL and its receptors. Young, P. R. (1997) J. Biol. Chem. 272, 14272–14276 18. Chinnaiyan, A. M., O’Rourke, K., Yu, G.-L., Lyons, R. H., Garg, M., Duan, Acknowledgments—We thank Jeremy Harrop, Kristy Kikly, Gordon D. R., Xing, L., Gentz, R., Ni, J., and Dixit, V. M. (1996) Science 274, Moore, Terence Porter, Ray Sweet, K. B. Tan, and Alem Truneh for 990 –992 helpful discussions, Preston Hensley, Sanjay Kumar, Steve Holmes, 19. Marsters, S. A., Sheridan, J. P., Donahue, C. J., Pitti, R. M., Gray, C. L., Josephine Fox, Tony Chadderton, Stefan Gluck, and K. B. Tan for Goddard, A. D., Bauer, K. D., and Ashkenazi, A. (1996) Curr. Biol. 6, sharing unpublished data, and Jennifer Shepard and Jeffrey Laydon for 1669 –1676 assistance with the osteoclastogenesis assay. 20. Kitson, J., Raven, T., Jiang, Y.-P., Goeddel, D. V., Giles, K. M., Pun, K.-T., Grinham, C. J., Brown, R., and Farrow, S. N. (1996) Nature 384, 372–375 21. Kumar, S., Minnich, M. D., and Young, P. R. (1995) J. Biol. Chem. 270, Note Added in Proof—Recently two groups (H. Yasuda et al. (1998) 27905–27913 Proc. Natl. Acad. Sci. U. S. A. 95, 3597–3602 and D. L. Lacey et al. 22. Johanson, K., Appelbaum, E., Doyle, M., Hensley, P., Zhao, B., Abdel-Meguid, (1998) Cell 93, 165–176) reported the identification of a different OPG S. S., Young, P., Cook, R., Carr, S., Matico, R., Cusimano, D., Dul, E., ligand that is identical to TRANCE/RANK-L. TL4 was recently de- Angelichio, M., Brooks, I., Winborne, E., McDonnell, P., Morton, T., scribed as LIGHT (D. N. Mauri et al. (1998) Immunity 8, 21–30), Bennett, D., Sokoloski, T., McNulty, D., Rosenberg, M., and Chaiken, I. another member of the TNF family. (1995) J. Biol. Chem. 270, 9459 –9471 23. Aiyar, N., Baker, E., Wu, H.-L., Nambi, P., Edwards, R. M., Trill, J. J., Ellis, REFERENCES C., and Bergsma, D. J. (1994) Mol. Cell. Biochem. 131, 75– 86 1. Lotz, M., Setareh, M., Kempis, J. V., and Schwarz, H. (1996) J. Leukocyte Biol. 24. Shatzman, A. R., and Rosenberg, M. (1987) Methods Enzymol. 152, 661– 673 60, 1–7 25. Harlow, E., and Lane, D. (1988) Antibodies: A laboratory Manual Cold Spring 2. Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Harbor Laboratory, Cold Spring Harbor, NY Ashkenazi, A. (1996) J. Biol. Chem. 271, 12687–12690 26. O’Shannessy, D. J., Brigham-Burke, M., and Peck, K. (1992) Anal. Biochem. 3. Nagata, S. (1997) Cell 88, 355–365 205, 132–136 4. Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C.-P., Nicholl, 27. O’Shannessy, D. J., Brigham-Burke, M., Soneson, K. K., Hensley, P., and J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and Goodwin, Brooks, I. (1994) Methods Enzymol. 240, 323–349 R. G. (1995) Immunity 3, 673– 682 28. Abu-Amer, Y., Ross, F. P., Edwards, J., and Teitelbaum, S. L. (1997) J. Clin. 5. Yuan, J. (1997) Curr. Opin. Cell Biol. 9, 247–251 Invest. 100, 1557–1565 6. Pan, G., O’Rourke, K., Chinnaiyan, A. M., Gentz, R., Ebner, R., Ni, J., and 29. Lacey, D. L., Erdmann, J. M., Teitelbaum, S. L., Tan, H.-L., Ohara, J., and Dixit, V. M. (1997) Science 276, 111–113 Shioi, A. (1995) Endocrinology 136, 2367–2376 7. Pan, G., Ni, J., Wei, Y.-F., Yu, G.-l., Gentz, R., and Dixit, V. M. (1997) Science 30. Smith, C. A., Gruss, H.-J., Davis, T., Anderson, D., Farrah, T., Baker, E., 277, 815– 818 Sutherland, G. R., Brannan, C. I., Copeland, N. G., Jenkins, N. A., 8. Sheridan, J. P., Marsters, S. A., Pitti, R. M., Gurney, A., Skubatch, M., Grabstein, K. H., Gliniak, B., McAlister, I. B., Fanslow, W., Alderson, M., Baldwin, D., Ramakrishnan, L., Gray, C. L., Baker, K., Wood, W. I., Falk, B., Gimpel, S., Gillis, S., Din, W. S., Goodwin, R. G., and Armitage, Goddard, A. D., Godowski, P., and Ashkenazi, A. (1997) Science 277, R. J. (1993) Cell 73, 1349 –1360 818 – 821 31. Pennica, D., Lam, V. T., Mize, N. K., Weber, R. F., Lewis, M., Fendly, B. M., 9. Schneider, P., Bodmer, J.-L., Thome, M., Hofmann, K., Holler, N., and Lipari, M. T., and Goeddel, D. V. (1992) J. Biol. Chem. 267, 21172–21178 Tschopp, J. (1997) FEBS Lett. 416, 329 –334 32. Loetscher, H., Gentz, R., Zulauf, M., Lustig, A., Tabuchi, H., Schlaeger, E. J., 10. MacFarlane, M., Ahmad, M., Srinivasula, S. M., Fernandes-Alnemri, T., Brockhaus, M., Gallati, H., Manneberg, M., and Lesslauer, W. (1991) Cohen, G., and Alnemri, E. S. (1997) J. Biol. Chem. 272, 25417–25420 J. Biol. Chem. 266, 18324 –18329 11. Screaton, G. R., Mongkolsapaya, J., Xu, X.-N., Cowper, A. E., McMichael, A. J., 33. Rothe, J., Gehr, G., Loetscher, H., and Lesslauer, W. (1992) Immunol. Res. 11, and Bell, J. I. (1997) Curr. Biol. 7, 693– 696 81–90 12. Walczak, H., Degli-Esposti, M. A., Johnson, R. S., Smolak, P. J., Waugh, J. Y., 34. Lewis, M., Tartaglia, L. A., Lee, A., Bennett, G. L., Rice, G. C., Wong, G. H., Boiani, N., Timour, M. S., Gerhart, M. J., Schooley, K. A., Smith, C. A., Chen, E. Y., and Goeddel, D. V. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, Goodwin, R. G., and Rauch, C. T. (1997) EMBO J. 16, 5386 –5397 2830 –2834 13. Marsters, S. A., Sheridan, J. P., Pitti, R. M., Huang, A., Skubatch, M., 35. Sedger, L., and McFadden, G. (1996) Immunol. Cell Biol. 74, 538 –545 Baldwin, D., Yuan, J., Gurney, A., Goddard, A. D., Godowski, P., and 36. Pinckard, J. K., Sheehan, K. C. F., Arthur, C. D., and Schreiber, R. D. (1997) Ashkenazi, A. (1997) Curr. Biol. 7, 1003–1006 J. Immunol. 158, 3869 –3873 14. Simonet, W. S., Lacey, D. L., Dunstan, C. R., Kelley, M., Chang, M.-S., Luthy, R., Nguyen, H. Q., Wooden, S., Bennett, L., Boone, T., Shimamoto, G., 37. Veis, D. J., Sorenson, C. M., Shutter, J. R., and Korsmeyer, S. J. (1993) Cell 75, 229 –240 DeRose, M., Elliot, R., Colombero, A., Tan, H.-L., Trail, G., Sullivan, J., http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Osteoprotegerin Is a Receptor for the Cytotoxic Ligand TRAIL

Download PDF
5 pages

Loading next page...
 
/lp/unpaywall/osteoprotegerin-is-a-receptor-for-the-cytotoxic-ligand-trail-IHUwdvFlVb

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
0021-9258
DOI
10.1074/jbc.273.23.14363
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 23, Issue of June 5, pp. 14363–14367, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. (Received for publication, January 26, 1998 and in revised form, March 9, 1998) John G. Emery‡, Peter McDonnell‡, Michael Brigham Burke¶, Keith C. Deeni, Sally Lyn‡, Carol Silverman**, Edward Dul‡‡, Edward R. Appelbaum‡‡, Chris Eichmani, Rocco DiPrinzioi, Robert A. Dodds§§, Ian E. James§§, Martin Rosenberg¶¶, John C. Lee§§, and Peter R. Young‡§ From the Departments of ‡Molecular Biology, ¶Structural Biology, iMolecular and Cellular Immunology, **Protein Biochemistry, ‡‡Gene Expression Sciences, and §§Bone and Cartilage Biology at SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406 and the ¶¶Department of Anti-infectives at SmithKline Beecham Pharmaceuticals, Collegeville, Pennsylvania 19426 TRAIL is a tumor necrosis factor-related ligand that member that increases bone density and causes splenomegaly when overexpressed in transgenic mice (14). OPG inhibits os- induces apoptosis upon binding to its death domain- containing receptors, DR4 and DR5. Two additional teoclastogenesis in vitro, suggesting that increased bone den- TRAIL receptors, TRID/DcR1 and DcR2, lack func- sity results from decreased numbers of mature osteoclasts. It tional death domains and function as decoy receptors has been suggested that OPG may neutralize a TNF-related for TRAIL. We have identified a fifth TRAIL receptor, ligand that promotes osteoclast differentiation or bind a mem- namely osteoprotegerin (OPG), a secreted tumor ne- brane-anchored TNF-related ligand that regulates osteoclasto- crosis factor receptor homologue that inhibits osteo- genesis via reverse signaling (14). clastogenesis and increases bone density in vivo. To identify interactions between novel TNF ligand and re- M, which OPG-Fc binds TRAIL with an affinity of 3.0 n ceptor family members, we screened a panel of receptor-Fc is slightly weaker than the interaction of TRID-Fc or fusion proteins for binding to TRAIL and have identified a DR5-Fc with TRAIL. OPG inhibits TRAIL-induced apo- novel interaction between OPG and TRAIL. We demonstrate ptosis of Jurkat cells. Conversely, TRAIL blocks the that OPG binds to TRAIL in vitro and can block TRAIL- anti-osteoclastogenic activity of OPG. These data sug- induced apoptosis. Conversely, TRAIL appears to block OPG- gest potential cross-regulatory mechanisms by OPG mediated inhibition of osteoclastogenesis in vitro. These results and TRAIL. suggest that OPG and TRAIL may function to inhibit each other. Members of the TNF ligand and receptor superfamilies play EXPERIMENTAL PROCEDURES an important role in regulating many biological processes, in- Preparation of Proteins and Antibodies—All cDNAs were identified cluding cytokine production, apoptosis, cell activation, lympho- by homology searches of an assembled Expressed Sequence Tag data cyte co-stimulation, proliferation, immunoglobulin secretion, base, and the 59-most cDNA clones were obtained. The cDNAs encoding and isotype switching (1). Three of the ligands, FasL, TNFa, the native leader peptides and extracellular domains of OPG (amino acids 1– 401) (14), herpes virus entry mediator (HVEM) (amino acids and TRAIL/Apo-2L, which are type II transmembrane proteins, 1–192) (15–17), DR3 (amino acids 1–199) (11, 18 –20), DR5 (amino acids exhibit potent cytotoxic activity, inducing apoptosis of suscep- 1–133 of Ref. 7) (7–12), and TRID (amino acids 1–240) (7–10) were tible cells within hours (2– 4). These cytotoxic ligands bind to polymerase chain reaction-amplified and subcloned upstream of an and aggregate type I transmembrane receptors with cytoplas- in-frame Factor Xa protease cleavage site and the hinge-Fc region of a mic “death domains,” which ultimately activate a protease cas- human IgG-g1 heavy chain in COSFclink (21, 22). The IL5R-Fc con- cade leading to apoptosis (5). struct was described previously (22). OPG-Fc, HVEM-Fc, DR3-Fc, and TRID-Fc were purified from conditioned medium of Chinese hamster Recently four receptors for TRAIL have been identified. Two ovary stable transfectants by protein G affinity chromatography (Am- of these receptors, DR4 (6) and DR5/TRICK2/TRAIL-R2 (7–12), ersham Pharmacia Biotech). Samples contaminated with Fc dimer were contain cytoplasmic death domains and are capable of inducing further purified on Superdex 200 (Amersham). DR5-Fc protein was apoptosis upon overexpression in transfected cells. Addition- purified from the conditioned medium of COS cell transient transfec- ally, two decoy receptors, DcR1/TRID/TRAIL-R3 (7–10) and tants by affinity chromatography on Prosep A (Bioprocessing Ltd.). DcR2 (13), lack functional death domains and inhibit TRAIL- Soluble receptors without the Fc domain were prepared by digesting with 1/80 e/s Factor Xa (Hematologic Technologies, Inc.) at 4 °C for 72 h. induced apoptosis. The existence of decoy receptors for TRAIL Fc dimer was removed by protein A affinity chromatography helps explain the absence of massive apoptosis in cells and (Amersham). tissues that express both TRAIL and one of its cytotoxic recep- Soluble TRAIL tagged at the N terminus with the FLAG epitope tors (7). However, in tissues lacking the decoy receptor, another (TRAIL-Flag, amino acids 95–281) (4) was constructed in the pCDN mechanism must exist to prevent TRAIL-induced apoptosis. mammalian expression vector (23) that had been modified to contain an Osteoprotegerin (OPG) is a secreted TNF receptor family in-frame tissue plasminogen activator signal sequence upstream of the FLAG-TRAIL coding region. Soluble TRAIL-Flag was purified from 30 liters of conditioned medium from a Chinese hamster ovary stable * The costs of publication of this article were defrayed in part by the transfectant by sequential chromatography on hydroxyapatite (Bio- payment of page charges. This article must therefore be hereby marked Rad, type I Macro Prep), Q-Sepharose, Mono Q, and Superdex 200 “advertisement” in accordance with 18 U.S.C. Section 1734 solely to (Amersham). TRAIL-Flag eluted at ;90 kDa, slightly larger than ex- indicate this fact. pected for a trimer, but appeared to be predominantly trimeric by § To whom correspondence should be addressed: Dept. of Molecular analytical ultracentrifugation analysis. Biology, Mail Code UW2101, SmithKline Beecham Pharmaceuticals, Polyclonal antibodies to TRAIL were raised in rabbits by injection of 709 Swedeland Rd., King of Prussia, PA 19406. Tel.: 610-270-7691; Fax: TRAIL (amino acids 41–281) that contained an epitope tag and hexa- 610-270-5114; E-mail: [email protected]. The abbreviations used are: TNF, tumor necrosis factor; OPG, os- teoprotegerin; ELISA, enzyme-linked immunosorbent assay; TRAP, tartrate-resistant acid phosphatase. P. Hensley, unpublished data. This paper is available on line at http://www.jbc.org 14363 This is an Open Access article under the CC BY license. 14364 Osteoprotegerin Is a Receptor for TRAIL histidine tag at its N terminus. The tagged TRAIL protein was ex- unlabeled TRAIL-Flag. Complexes were captured with 30 ml of protein pressed in Escherichia coli as insoluble inclusion bodies using the pAS A-Sepharose 4B, washed twice in binding buffer, and electrophoresed expression system (24), solubilized in guanidine HCl, and purified by through 10% SDS-polyacrylamide gel electrophoresis. Gels were fixed nickel nitrilotriacetic acid chromatography. for 30 min, incubated with Enlighten (Amersham) for 30 min, and dried Receptor Precipitations—TRAIL-Flag (250 ng) was added to 2 mgof before autoradiography. receptor-Fc in binding buffer (25 mM HEPES, pH 7.2, 0.25% bovine ELISAs—ELISAs were performed according to standard techniques serum albumin, 0.01% Tween in RPMI 1640) and incubated for2hon (25). Immunlon 4 (Dynatech Laboratories) plates were coated overnight ice. Protein A-Sepharose 4B (Amersham, 30 ml of a 75% slurry) was at 4 °C with anti-Flag M2 monoclonal antibody (Eastman Kodak Co.) (2 added and incubated for1hon ice. Complexes were recovered by mg/ml, 100 ml/well) in 0.05 M bicarbonate buffer, pH 8.6. After washing centrifugation, washed three times with binding buffer, electrophoresed and blocking with 0.5% gelatin in phosphate-buffered saline, TRAIL- on 15% SDS-polyacrylamide gel electrophoresis, and transferred to Flag (2 mg/ml, 100 ml/well) was bound for1hat room temperature. nitrocellulose for Western analysis with an anti-TRAIL polyclonal an- Serial dilutions of OPG-Fc or DR5-Fc (1 mg/ml to 5 ng/ml, 100 ml/well) tiserum. For binding reactions with full-length TRAIL, the entire were added after washing and incubated for1hat room temperature. TRAIL coding region was polymerase chain reaction-amplified and The Fc fusions were detected with a biotinylated goat anti-human IgG cloned into pCDN (23). HEK293 cells (3 3 10 ) were transfected with 10 antibody (Southern Biotechnology Associates) (1/2000 dilution, 100 ml/ mg of pCDN-TRAIL or pCDN alone using lipofectAMINE reagent (Life well), horseradish peroxidase-conjugated streptavidin (Southern Bio- Technologies, Inc.). 48 h after transfection, regular growth medium was technology Associates) (1/4000 dilution, 100 ml/well), and ABTS perox- replaced with methionine/cysteine-free Dulbecco’s modified Eagle’s me- idase substrate (100 ml/well, Kirkegaard and Perry Laboratories). The dium. After a 1-h starvation, 1.5 mCi of translabel [ S]Met,Cys (ICN) was added to transfected cells along with 2% dialyzed fetal bovine TABLE I serum for 6 h. Cells were washed in Dulbecco’s modified Eagle’s me- Binding constants for the interaction of TRAIL-Flag with OPG-Fc, dium and lysed in 1 ml of STET (150 mM NaCl, 10 mM Tris, pH 7.5, 1 TRID-Fc, and DR5-Fc mM EDTA, 1% Triton X-100, 10 mg/ml leupeptin, 20 mg/ml aprotinin, 2 Receptor k k Calculated K a d D mM phenylmethylsulfonyl fluoride) for 20 min on ice. Lysates were 21 21 21 centrifuged, and the supernatants were precleared with 100 ml of pro- M s s nM tein A-Sepharose 4B (Amersham). 50 ml of the precleared lysate was 4 24 OPG-Fc 6.6 3 10 2.0 3 10 3.0 used in receptor precipitation with 2 mg of OPG-Fc as described above. 5 24 TRID-Fc 2.9 3 10 3.2 3 10 1.1 Binding volumes were increased to 1 ml, and the binding reaction 5 24 DR5-Fc 3.4 3 10 2.6 3 10 0.76 proceeded overnight at 4 °C. Competition was achieved with 2 mgof FIG.1. OPG binds to TRAIL. A, immunoprecipitation of TRAIL-Flag by receptor-Fc proteins. TRAIL-Flag was incubated with the indicated receptor-Fc proteins, and complexes were precipitated with protein A-Sepharose. The samples were analyzed by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose for Western analysis with an anti-TRAIL polyclonal antiserum. B, cleaved OPG, TRID, and DR5 compete with Fc fusions for binding to TRAIL. Binding reactions were essentially as described above, except that a 10-fold molar excess of cleaved receptor was added immediately before the Fc fusion. C, OPG-Fc binds to cell-associated full-length TRAIL (FL-TRAIL). 293 cells were transiently transfected with a full-length TRAIL expression construct and labeled with [ S]methionine, and cell lysates were subject to immunoprecipitation. Unlabeled soluble TRAIL-Flag was added as above where indicated. D, OPG- and DR5-Fc bind to TRAIL-Flag in ELISA. Microtiter plates were coated with an anti-Flag M2 monoclonal antibody and allowed to bind TRAIL-Flag. The plates were incubated with serial dilutions of OPG-Fc (M) and DR5-Fc (l ) proteins, and binding was detected with a biotinylated goat anti-human IgG antibody followed by streptavidin-horseradish peroxidase and ABTS peroxidase substrate. E, OPG and DR5 compete with OPG-Fc for binding to TRAIL-Flag. The assay was performed essentially as described above, except that serial dilutions of cleaved competitor OPG (M) or DR5 (l ) were incubated with the anti-Flag-captured TRAIL-Flag before addition of OPG-Fc. Samples without competitor are indicated by ‚. s, soluble; KD, kilodaltons. Osteoprotegerin Is a Receptor for TRAIL 14365 FIG.2. OPG blocks TRAIL-induced apoptosis. Jurkat cells were treated with increasing amounts of OPG (A), DR5-Fc (B), or DR3-Fc (C) in the presence of TRAIL-Flag plus anti-FLAG M2 mono- clonal antibody (Kodak) or the anti-Fas agonist monoclonal antibody CH-11 (MBL) for 3 h. Cytotoxicity was assessed by de- termining the percentage of propidium iodide (PI)-permeable cells (mean 6 S.D., n 5 2) by fluorescence-activated cell sorter analysis. D and E, cytospins of Jurkat cells treated with TRAIL-Flag as described above in the absence (D)or presence (E) of OPG (5 mg/ml) were stained with 49,6-diamidine-29-phenylin- dole dihydrochloride, and the nuclei were examined by fluorescence microscopy. Note the dramatic reduction in nuclear fragmentation in OPG-treated cells. plates were read at 405 nm on a UV max microplate reader (Molecular 7 days, after which the media were removed, and the cell monolayer Devices). For competition assays, serial dilutions of OPG or DR5 was stained for tartrate-resistant acid phosphatase (TRAP) or by a without Fc domains (50 mg/ml to 30 ng/ml, 100 ml/well) were added to TRAP-solution assay (29). In the TRAP-solution assay, enzyme activity the plate for1hat room temperature before the addition of OPG-Fc (10 was measured by the conversion of p-nitrophenylphosphate (20 nM)to mg/ml, 10 ml/well). p-nitrophenol in the presence of 80 mM sodium tartrate and expressed Determination of the Affinity of TRAIL for Its Receptors—The as- as OD405 units. sociation and dissociation rates of the interaction of TRAIL with RESULTS AND DISCUSSION captured receptor fusion proteins were determined by surface plas- mon resonance using a BIAcore 1000 (BIAcore Inc.). The capture To identify interactions between novel TNF ligand and re- surface was a protein A (Pierce) modified CM5 sensor chip (26). The ceptor family members, we screened a panel of receptor-Fc sensor surface was equilibrated with a buffer of 20 mM sodium phos- fusion proteins for binding to TRAIL by immunoprecipitation. phate, 150 mM sodium chloride, and 0.005% Tween 20, pH 7.4, and TRAIL bound to TRID-Fc and DR5-Fc proteins as expected analyses were performed at 30 ml/min at 25 °C. The receptor-Fc (Fig. 1A). TRAIL also bound to OPG-Fc. TRAIL did not bind to fusion protein was diluted into the above buffer to 1 mg/ml, and a 20 ml injection was passed over the capture surface, followed by a 150-ml DR3-Fc and HVEM-Fc (Fig. 1A), two additional members of the injection of the TRAIL-Flag. After the association phase, 500 s of TNF receptor superfamily, indicating that the OPG-TRAIL dissociation data was collected. The surface was regenerated after interaction is not Fc-mediated. To confirm that binding of each cycle. Sets of 3– 4 analyte concentrations, 500 20 nM, were OPG-Fc to TRAIL was not to the Fc region, a 10-fold molar collected and analyzed by nonlinear regression analysis (27) using the excess of OPG cleaved of its Fc domain was incubated with BIAevaluation software 2.1. The dissociation data was fitted on the TRAIL and the Fc fusion proteins before immunoprecipitation. basis of the AB 5 A 1 B model. The association data was fitted to A 1 B 5 AB using the type 1 model. Cleaved OPG, TRID, and DR5 effectively competed away the Apoptosis Assays—Jurkat cells (ATCC) (5 3 10 cells/ml) were binding of OPG-Fc to TRAIL (Fig. 1B). To determine if OPG treated with increasing concentrations of OPG (0, 0.2, 1.0, or 5.0 mg/ml), could bind to cell-associated TRAIL, HEK293 cells were tran- DR5-Fc (0, 0.008, 0.04, and 0.2 mg/ml), or DR3-Fc (0, 0.2, 1.0, or 5.0 siently transfected with a full-length TRAIL expression vector, mg/ml), and apoptosis was induced with an anti-Fas CH-11 monoclonal labeled with [ S]methionine, and the cell lysate was subjected antibody (0.1 mg/ml, Medical and Biological Laboratories) or TRAIL- to immunoprecipitation by the OPG-Fc fusion protein. OPG-Fc Flag (0.1 mg/ml) with anti-FLAG M2 monoclonal antibody (3 mg/ml, Kodak) for 3 h. The anti-Flag M2 antibody was added to all treatments precipitated a protein of the expected size in TRAIL but not to normalize for potential nonspecific effects. Cells were stained with vector-transfected cells (Fig. 1C), indicating that OPG-Fc binds propidium iodide (6.5 mg/ml, Boehringer Mannheim) and quantitated to cell-associated TRAIL. Unlabeled soluble TRAIL-Flag effec- by fluorescence-activated cell sorter analysis. Cytospins of Jurkat cells tively competed with the S-labeled full-length TRAIL for were fixed for 30 min in ice-cold 1% paraformaldehyde in phosphate- binding to OPG-Fc. buffered saline, washed in phosphate-buffered saline, and stained for The ability of TRAIL to bind OPG was also examined by 30 min with 49,6-diamidine-29-phenylindole dihydrochloride (1 mg/ml, Boehringer Mannheim). ELISA. Both OPG-Fc and DR5-Fc proteins were capable of Osteoclastogenesis Assays—A modified osteoclast formation assay binding to anti-Flag-immobilized TRAIL-Flag (Fig. 1D). In was adapted from Ref. 28. In brief, bone marrow mononuclear cells were blocking assays, cleaved DR5 prevented OPG-Fc from binding isolated from femurs of 4 – 6-week DBF1 mice in the presence of early to TRAIL-Flag (Fig. 1E), indicating that the DR5 and OPG passage stromal cells obtained from a human osteoclastoma at a 400:1 binding sites overlap. Cleaved OPG was more effective than ratio. The co-culture was established in RPMI 1640 supplemented with cleaved DR5 in blocking TRAIL-Flag binding (Fig. 1E), possibly 10% fetal calf serum, 100 nM dexamethasone, 10 nM vitamin D3. Test reagents were added at day 0, and the cultures were left undisturbed for reflecting the fact that the cleaved OPG is a dimer, whereas 14366 Osteoprotegerin Is a Receptor for TRAIL Anti-Flag-aggregated TRAIL-Flag exhibited significant cyto- toxicity within 3 h (Fig. 2A). OPG demonstrated a dose-depend- ent inhibition of TRAIL-induced cytotoxicity and blocked the appearance of apoptotic nuclei in Jurkat cells treated with TRAIL-Flag (Fig. 2, D and E). However, OPG did not prevent killing by an agonist anti-Fas monoclonal antibody, another potent inducer of apoptosis, indicating that OPG does not ac- tivate a nonspecific survival pathway (Fig. 2A). DR5-Fc exhib- ited a similar but more potent inhibition of TRAIL-induced cytotoxicity (Fig. 2B), likely due to its higher affinity for TRAIL. DR3-Fc, which does not bind TRAIL, did not inhibit TRAIL-induced killing (Fig. 2C). This data suggests that OPG, which is secreted and detectable in the circulation (14), may be a soluble antagonist receptor for TRAIL. OPG may negatively regulate TRAIL activity, similar to the inhibition of TNFa by secreted TNF receptors of poxviruses (35). Furthermore, TNF receptors are shed from the cell surface and found in the circulation following certain stimuli. It has been suggested that soluble TNF receptors may inhibit or clear circulating TNFa (36). A similar role for OPG might also underlie the splenomeg- aly in mice that overexpress OPG (14). In mice lacking the anti-apoptotic Bcl-2 gene, there is a decrease in the size of the spleen, suggesting that spleen cell homeostasis is regulated by apoptotic mechanisms (37). Thus, if TRAIL is involved in reg- ulation of spleen cell homeostasis by inducing apoptosis, then constitutive overexpression of OPG might result in decreased apoptosis and splenomegaly. OPG inhibits osteoclastogenesis in vitro (14). To determine the significance of the OPG-TRAIL interaction in osteoclasto- genesis, we examined the effects of TRAIL in an in vitro osteo- clastogenesis assay. Human stromal cells were co-cultured with murine bone marrow cells for 7 days in the presence of OPG, TRAIL, or TL4, another TNF-like ligand that does not bind OPG. OPG inhibited the formation of multinucleate TRAP positive osteoclasts as expected, with an IC of approx- imately 60 pM (Fig. 3). TRAIL alone had no effect on osteoclas- togenesis. However, TRAIL completely blocked the inhibitory effect of OPG (Fig. 3), suggesting that soluble TRAIL may function to regulate the activity of OPG. TL4 had no effect on osteoclastogenesis in the presence or absence of OPG. It has been suggested that OPG may inhibit osteoclastogen- esis by binding to a pro-osteoclastogenic TNF-related ligand (14). Our data suggest that this ligand may not be TRAIL. First, soluble TRAIL alone had no effect on osteoclastogenesis, FIG.3. TRAIL blocks the ability of OPG to inhibit osteoclasto- and the IC of OPG is approximately 50-fold lower than the genesis. In vitro osteoclastogenesis assays in the absence (A, C, and E) affinity of OPG for soluble TRAIL. Secondly, anti-Flag-aggre- or presence of OPG (10 ng/ml) (B, D,, and F), TRAIL-Flag (C and D), or gated TRAIL, which may mimic membrane TRAIL, induced TL4 (E and F), a novel TNF homologue that does not bind OPG. Arrows massive apoptosis in these co-cultures, which is the opposite in A indicate the multinucleate TRAP-positive osteoclasts. G, quanti- activity to what one would expect for the OPG ligand (data not tation of TRAP levels in osteoclastogenesis assay. shown). However, further experiments will be necessary to cleaved DR5 is a monomer (Ref. 14 and data not shown). fully define the role of TRAIL in osteoclastogenesis and the Although overlapping, the binding sites of OPG-Fc and DR5-Fc existence of other OPG ligands. are not identical, since only OPG-Fc recognized directly immo- The identification of a fifth receptor for TRAIL suggests that bilized TRAIL-Flag (i.e. without an anti-Flag antibody, data complex regulatory mechanisms control its activity. A soluble not shown). antagonist receptor, OPG, and two decoy receptors, DcR1/ The affinity of each receptor for TRAIL was determined by TRID/TRAIL-R3 and DcR2, may prevent the transduction of measuring the kinetics of TRAIL binding to each by surface apoptotic signaling through the death receptors DR4 and DR5/ plasmon resonance (Table I). OPG-Fc binds to TRAIL-Flag TRICK2/TRAIL-R2. It has also been suggested that the gener- with an affinity of 3.0 nM, which is slightly weaker than the ation of two DR5/TRICK2/TRAIL-R2 isoforms by alternate affinities of TRID-Fc and DR5-Fc for TRAIL-Flag (1.1 nM and splicing may regulate cellular responses to TRAIL (11). Addi- 0.76 nM, respectively). The affinities of each receptor are within the range of affinities reported for other physiologically rele- S. Kumar, S. Holmes, J. Fox, S. Gluck, T. Chadderton, and K. B. vant TNF-like ligand and receptor pairs (30 –34). Tan, unpublished data. The observation that OPG binds TRAIL in vitro suggests J. A. Harrop, P. C. McDonnell, M. Brigham-Burke, S. D. Lyn, J. that OPG may inhibit the cytotoxic activity of TRAIL. To ex- Minton, K. B. Tan, K. Dede, J. Spampanato, C. Silverman, P. Hensley, amine this possibility, Jurkat cells were treated with anti-Flag- R. DiPrinzio, J. G. Emery, C. Eichman, M. Chabot-Fletcher, A. Truneh, aggregated TRAIL-Flag in the presence or absence of OPG. and P. R. Young, submitted for publication. Osteoprotegerin Is a Receptor for TRAIL 14367 Davy, E., Bucay, N., Renshaw-Gegg, L., Hughes, T. M., Hill, D., Pattison, tionally, secreted TRAIL itself may antagonize OPG activity or W., Campbell, P., Sander, S., Van, G., Tarpley, J., Derby, P., Lee, R., prevent signaling by membrane-anchored TRAIL through its Program, A. E., and Boyle, W. J. (1997) Cell 89, 309 –319 death receptors, as has been described for FasL and TNFa 15. Montgomery, R. I., Warner, M. S., Lum, B. J., and Spear, P. G. (1996) Cell 87, 427– 436 (reviewed in Ref. 3). A complete understanding of these regu- 16. Hsu, H., Solovyev, I., Colombero, A., Elliott, R., Kelley, M., and Boyle, W. J. latory circuits awaits the determination of soluble TRAIL and (1997) J. Biol. Chem. 272, 13471–13474 OPG levels in vivo and an analysis of knockout mutants of 17. Kwon, B. S., Tan, K. B., Ni, J., Kim, K. K., Kim, Y.-J., Wang, S., Gentz, R., Yu, G.-L., Harrop, J., Lyn, S. D., Silverman, C., Porter, T. G., Truneh, A., and TRAIL and its receptors. Young, P. R. (1997) J. Biol. Chem. 272, 14272–14276 18. Chinnaiyan, A. M., O’Rourke, K., Yu, G.-L., Lyons, R. H., Garg, M., Duan, Acknowledgments—We thank Jeremy Harrop, Kristy Kikly, Gordon D. R., Xing, L., Gentz, R., Ni, J., and Dixit, V. M. (1996) Science 274, Moore, Terence Porter, Ray Sweet, K. B. Tan, and Alem Truneh for 990 –992 helpful discussions, Preston Hensley, Sanjay Kumar, Steve Holmes, 19. Marsters, S. A., Sheridan, J. P., Donahue, C. J., Pitti, R. M., Gray, C. L., Josephine Fox, Tony Chadderton, Stefan Gluck, and K. B. Tan for Goddard, A. D., Bauer, K. D., and Ashkenazi, A. (1996) Curr. Biol. 6, sharing unpublished data, and Jennifer Shepard and Jeffrey Laydon for 1669 –1676 assistance with the osteoclastogenesis assay. 20. Kitson, J., Raven, T., Jiang, Y.-P., Goeddel, D. V., Giles, K. M., Pun, K.-T., Grinham, C. J., Brown, R., and Farrow, S. N. (1996) Nature 384, 372–375 21. Kumar, S., Minnich, M. D., and Young, P. R. (1995) J. Biol. Chem. 270, Note Added in Proof—Recently two groups (H. Yasuda et al. (1998) 27905–27913 Proc. Natl. Acad. Sci. U. S. A. 95, 3597–3602 and D. L. Lacey et al. 22. Johanson, K., Appelbaum, E., Doyle, M., Hensley, P., Zhao, B., Abdel-Meguid, (1998) Cell 93, 165–176) reported the identification of a different OPG S. S., Young, P., Cook, R., Carr, S., Matico, R., Cusimano, D., Dul, E., ligand that is identical to TRANCE/RANK-L. TL4 was recently de- Angelichio, M., Brooks, I., Winborne, E., McDonnell, P., Morton, T., scribed as LIGHT (D. N. Mauri et al. (1998) Immunity 8, 21–30), Bennett, D., Sokoloski, T., McNulty, D., Rosenberg, M., and Chaiken, I. another member of the TNF family. (1995) J. Biol. Chem. 270, 9459 –9471 23. Aiyar, N., Baker, E., Wu, H.-L., Nambi, P., Edwards, R. M., Trill, J. J., Ellis, REFERENCES C., and Bergsma, D. J. (1994) Mol. Cell. Biochem. 131, 75– 86 1. Lotz, M., Setareh, M., Kempis, J. V., and Schwarz, H. (1996) J. Leukocyte Biol. 24. Shatzman, A. R., and Rosenberg, M. (1987) Methods Enzymol. 152, 661– 673 60, 1–7 25. Harlow, E., and Lane, D. (1988) Antibodies: A laboratory Manual Cold Spring 2. Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Harbor Laboratory, Cold Spring Harbor, NY Ashkenazi, A. (1996) J. Biol. Chem. 271, 12687–12690 26. O’Shannessy, D. J., Brigham-Burke, M., and Peck, K. (1992) Anal. Biochem. 3. Nagata, S. (1997) Cell 88, 355–365 205, 132–136 4. Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C.-P., Nicholl, 27. O’Shannessy, D. J., Brigham-Burke, M., Soneson, K. K., Hensley, P., and J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and Goodwin, Brooks, I. (1994) Methods Enzymol. 240, 323–349 R. G. (1995) Immunity 3, 673– 682 28. Abu-Amer, Y., Ross, F. P., Edwards, J., and Teitelbaum, S. L. (1997) J. Clin. 5. Yuan, J. (1997) Curr. Opin. Cell Biol. 9, 247–251 Invest. 100, 1557–1565 6. Pan, G., O’Rourke, K., Chinnaiyan, A. M., Gentz, R., Ebner, R., Ni, J., and 29. Lacey, D. L., Erdmann, J. M., Teitelbaum, S. L., Tan, H.-L., Ohara, J., and Dixit, V. M. (1997) Science 276, 111–113 Shioi, A. (1995) Endocrinology 136, 2367–2376 7. Pan, G., Ni, J., Wei, Y.-F., Yu, G.-l., Gentz, R., and Dixit, V. M. (1997) Science 30. Smith, C. A., Gruss, H.-J., Davis, T., Anderson, D., Farrah, T., Baker, E., 277, 815– 818 Sutherland, G. R., Brannan, C. I., Copeland, N. G., Jenkins, N. A., 8. Sheridan, J. P., Marsters, S. A., Pitti, R. M., Gurney, A., Skubatch, M., Grabstein, K. H., Gliniak, B., McAlister, I. B., Fanslow, W., Alderson, M., Baldwin, D., Ramakrishnan, L., Gray, C. L., Baker, K., Wood, W. I., Falk, B., Gimpel, S., Gillis, S., Din, W. S., Goodwin, R. G., and Armitage, Goddard, A. D., Godowski, P., and Ashkenazi, A. (1997) Science 277, R. J. (1993) Cell 73, 1349 –1360 818 – 821 31. Pennica, D., Lam, V. T., Mize, N. K., Weber, R. F., Lewis, M., Fendly, B. M., 9. Schneider, P., Bodmer, J.-L., Thome, M., Hofmann, K., Holler, N., and Lipari, M. T., and Goeddel, D. V. (1992) J. Biol. Chem. 267, 21172–21178 Tschopp, J. (1997) FEBS Lett. 416, 329 –334 32. Loetscher, H., Gentz, R., Zulauf, M., Lustig, A., Tabuchi, H., Schlaeger, E. J., 10. MacFarlane, M., Ahmad, M., Srinivasula, S. M., Fernandes-Alnemri, T., Brockhaus, M., Gallati, H., Manneberg, M., and Lesslauer, W. (1991) Cohen, G., and Alnemri, E. S. (1997) J. Biol. Chem. 272, 25417–25420 J. Biol. Chem. 266, 18324 –18329 11. Screaton, G. R., Mongkolsapaya, J., Xu, X.-N., Cowper, A. E., McMichael, A. J., 33. Rothe, J., Gehr, G., Loetscher, H., and Lesslauer, W. (1992) Immunol. Res. 11, and Bell, J. I. (1997) Curr. Biol. 7, 693– 696 81–90 12. Walczak, H., Degli-Esposti, M. A., Johnson, R. S., Smolak, P. J., Waugh, J. Y., 34. Lewis, M., Tartaglia, L. A., Lee, A., Bennett, G. L., Rice, G. C., Wong, G. H., Boiani, N., Timour, M. S., Gerhart, M. J., Schooley, K. A., Smith, C. A., Chen, E. Y., and Goeddel, D. V. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, Goodwin, R. G., and Rauch, C. T. (1997) EMBO J. 16, 5386 –5397 2830 –2834 13. Marsters, S. A., Sheridan, J. P., Pitti, R. M., Huang, A., Skubatch, M., 35. Sedger, L., and McFadden, G. (1996) Immunol. Cell Biol. 74, 538 –545 Baldwin, D., Yuan, J., Gurney, A., Goddard, A. D., Godowski, P., and 36. Pinckard, J. K., Sheehan, K. C. F., Arthur, C. D., and Schreiber, R. D. (1997) Ashkenazi, A. (1997) Curr. Biol. 7, 1003–1006 J. Immunol. 158, 3869 –3873 14. Simonet, W. S., Lacey, D. L., Dunstan, C. R., Kelley, M., Chang, M.-S., Luthy, R., Nguyen, H. Q., Wooden, S., Bennett, L., Boone, T., Shimamoto, G., 37. Veis, D. J., Sorenson, C. M., Shutter, J. R., and Korsmeyer, S. J. (1993) Cell 75, 229 –240 DeRose, M., Elliot, R., Colombero, A., Tan, H.-L., Trail, G., Sullivan, J.,

Journal

Journal of Biological Chemistry – Unpaywall

Published: Jun 1, 1998

There are no references for this article.