FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2019905662> ?p ?o ?g. }

• W2019905662 endingPage "23921" @default.
• W2019905662 startingPage "23915" @default.
• W2019905662 abstract "CD47 is a surface receptor that induces either coactivation or apoptosis in lymphocytes, depending on the ligand(s) bound. Interestingly, the apoptotic pathway is independent of caspase activation and cytochrome c release and is accompanied by early mitochondrial dysfunction with suppression of mitochondrial membrane potential (Δψm). Using CD47 as bait in a yeast two-hybrid system, we identified the Bcl-2 homology 3 (BH3)-only protein 19 kDa interacting protein-3 (BNIP3), a pro-apoptotic member of the Bcl-2 family, as a novel partner. Interaction between CD47 and the BH3-only protein was confirmed by immunoprecipitation analysis, and CD47-induced apoptosis was inhibited by attenuating BNIP3 expression with antisense oligonucleotides. Finally, we showed that the C-terminal domain of thrombospondin-1 (TSP-1), but not signal-regulatory protein (SIRPα1), is the ligand for CD47 involved in inducing cell death. Immunofluorescence analysis of CD47 and BNIP3 revealed a partial colocalization of both molecules under basal conditions. After T cell stimulation via CD47, BNIP3 translocates to the mitochondria to induce apoptosis. These results show that the BH3-dependent apoptotic pathways, previously shown to be activated by intracellular pro-apoptotic events, can also be turned on by surface receptors. This new pathway results in a fast induction of cell death resembling necrosis, which is likely to play an important role in lymphocyte regulation at inflammatory sites and/or in the vicinity of thrombosis. CD47 is a surface receptor that induces either coactivation or apoptosis in lymphocytes, depending on the ligand(s) bound. Interestingly, the apoptotic pathway is independent of caspase activation and cytochrome c release and is accompanied by early mitochondrial dysfunction with suppression of mitochondrial membrane potential (Δψm). Using CD47 as bait in a yeast two-hybrid system, we identified the Bcl-2 homology 3 (BH3)-only protein 19 kDa interacting protein-3 (BNIP3), a pro-apoptotic member of the Bcl-2 family, as a novel partner. Interaction between CD47 and the BH3-only protein was confirmed by immunoprecipitation analysis, and CD47-induced apoptosis was inhibited by attenuating BNIP3 expression with antisense oligonucleotides. Finally, we showed that the C-terminal domain of thrombospondin-1 (TSP-1), but not signal-regulatory protein (SIRPα1), is the ligand for CD47 involved in inducing cell death. Immunofluorescence analysis of CD47 and BNIP3 revealed a partial colocalization of both molecules under basal conditions. After T cell stimulation via CD47, BNIP3 translocates to the mitochondria to induce apoptosis. These results show that the BH3-dependent apoptotic pathways, previously shown to be activated by intracellular pro-apoptotic events, can also be turned on by surface receptors. This new pathway results in a fast induction of cell death resembling necrosis, which is likely to play an important role in lymphocyte regulation at inflammatory sites and/or in the vicinity of thrombosis. Multicellular organisms eliminate excess, damaged or infected cells by stereotypic programs of cell death (PCD). 1The abbreviations used are: PCD, programmed cell death; TSP-1, thrombospondin-1; SIRPα1, signal-regulatory protein; IAP, integrin-associated protein; TM, transmembrane; BNIP3, 19 kDa interacting protein-3; BH3, Bcl-2 homology 3; GFP, green fluorescent protein; JIN, Jurkat IAP-negative cells; Δψm, mitochondrial membrane potential; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; TOM, translocase of outer membrane; MMS, multiply membrane spanning; CD47ec, CD47 extracellular; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate. 1The abbreviations used are: PCD, programmed cell death; TSP-1, thrombospondin-1; SIRPα1, signal-regulatory protein; IAP, integrin-associated protein; TM, transmembrane; BNIP3, 19 kDa interacting protein-3; BH3, Bcl-2 homology 3; GFP, green fluorescent protein; JIN, Jurkat IAP-negative cells; Δψm, mitochondrial membrane potential; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; TOM, translocase of outer membrane; MMS, multiply membrane spanning; CD47ec, CD47 extracellular; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate. In its classic form, apoptosis is characterized by well defined ultrastructural changes including cell shrinkage, exposure of phosphatidylserine at the outer leaflet of the cytoplasmic membrane, changes in mitochondrial permeability, membrane blebbing, caspases activation, and DNA degradation. Lymphocyte PCD plays an important role in controlling immune responses and occurs both in central and peripheral lymphoid organs. Disturbed PCD may contribute to multiple immune disorders such as cancer and autoimmune and degenerative diseases. Upon signaling, pathways that influence T cell proliferation and survival, CD95/CD95L and tumor necrosis factor receptor pathways have been extensively studied over the past few years (1Siegel R.M. Chan F.K. Chun H.J. Lenardo M.J. Nat. Immunol. 2000; 1: 469-474Google Scholar). However, a number of others T cell surface receptors such as major histocompatibility complex class I and II (2Skov S. Klausen P. Claesson M.H. J. Cell Biol. 1997; 139: 1523-1531Google Scholar), CD2 (3Deas O. Dumont C. MacFarlane M. Rouleau M. Hebib C. Harper F. Hirsch F. Charpentier B. Cohen G.M. Senik A. J. Immunol. 1998; 161: 3375-3383Google Scholar), CD4 (4Berndt C. Mopps B. Angermuller S. Gierschik P. Krammer P.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12556-12561Google Scholar), CD45 (5Lesage S. Steff A.M. Philippoussis F. Page M. Trop S. Mateo V. Hugo P. J. Immunol. 1997; 159: 4762-4771Google Scholar, 6Klaus S.J. Sidorenko S.P. Clark E.A. J. Immunol. 1996; 156: 2743-2753Google Scholar), or CD99 (7Bernard G. Breittmayer J.P. de Matteis M. Trampont P. Hofman P. Senik A. Bernard A. J. Immunol. 1997; 158: 2543-2550Google Scholar) can also trigger PCD. In contrast to the former pathways, they have all been described to act independently of any of the known caspases (8Scaffidi C. Kirchhoff S. Krammer P.H. Peter M.E. Curr. Opin. Immunol. 1999; 11: 277-285Google Scholar), and the molecular mechanisms and the physiological and/or the pathological relevance of these death pathways remains to be established. Among these molecules, recent reports implicate CD47 in the triggering of atypical cell death (9Pettersen R.D. Hestdal K. Olafsen M.K. Lie S.O. Lindberg F.P. J. Immunol. 1999; 162: 7031-7040Google Scholar, 10Mateo V. Lagneaux L. Bron D. Biron G. Armant M. Delespesse G. Sarfati M. Nat. Med. 1999; 5: 1277-1284Google Scholar). CD47 (also known as IAP for integrin-associated protein), expressed on all mammalian cells (11Brown E. Hooper L. Ho T. Gresham H. J. Cell Biol. 1990; 111: 2785-2794Google Scholar), displays an extracellular Ig-like domain with five transmembrane (TM) segments and a short C-terminal cytoplasmic tail. CD47 is associated with β3 integrins on several cell types. However, on other cell types such as lymphocytes, no association with integrins has been documented. More generally, CD47 has been shown to activate integrins, either through direct interaction or at a distance (11Brown E. Hooper L. Ho T. Gresham H. J. Cell Biol. 1990; 111: 2785-2794Google Scholar, 12Lindberg F.P. Gresham H.D. Schwarz E. Brown E.J. J. Cell Biol. 1993; 123: 485-496Google Scholar, 13Brittain J.E. Mlinar K.J. Anderson C.S. Orringer E.P. Parise L.V. J. Clin. Invest. 2001; 107: 1555-1562Google Scholar). The two natural ligands currently known for CD47 are thrombospondin-1 (TSP-1), a protein found in extracellular matrix and released in large amounts by platelets upon activation, and the signal-regulatory protein (SIRPα1), expressed on the surface of macrophages and endothelial and dendritic cells. Moreover, we and others have shown that CD47 can trigger T cell activation and proliferation (14Ticchioni M. Deckert M. Mary F. Bernard G. Brown E.J. Bernard A. J. Immunol. 1997; 158: 677-684Google Scholar, 15Reinhold M.I. Lindberg F.P. Kersh G.J. Allen P.M. Brown E.J. J. Exp. Med. 1997; 185: 1-11Google Scholar, 16Waclavicek M. Majdic O. Stulnig T. Berger M. Baumruker T. Knapp W. Pickl W.F. J. Immunol. 1997; 159: 5345-5354Google Scholar) or induce T cell spreading (17Reinhold M.I. Green J.M. Lindberg F.P. Ticchioni M. Brown E.J. Int. Immunol. 1999; 11: 707-718Google Scholar). Therefore an important task has been to determine under which condition(s) CD47 induces T cell death and/or survival. Indeed, in addition to the cytoplasmic proteins Gi (18Frazier W.A. Gao A.G. Dimitry J. Chung J. Brown E.J. Lindberg F.P. Linder M.E. J. Biol. Chem. 1999; 274: 8554-8560Google Scholar, 19Gao A.G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Google Scholar) and proteins linking IAP with cytoskeleton (PLIC) (20Wu A.L. Wang J. Zheleznyak A. Brown E.J. Mol. Cell. 1999; 4: 619-625Google Scholar), we show in the present study that CD47 associates with the pro-apoptotic molecule 19 kDa interacting protein-3 (BNIP3). BNIP3 belongs to the Bcl-2 homology 3 (BH3)-only family, a Bcl-2-related family possessing an atypical Bcl-2 homology 3 (BH3) domain, which regulates PCD from mitochondrial sites by selective Bcl-2/Bcl-XL interactions (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar, 22Yasuda M. Theodorakis P. Subramanian T. Chinnadurai G. J. Biol. Chem. 1998; 273: 12415-12421Google Scholar). BNIP3 family members contain a C-terminal transmembrane domain that is required for their mitochondrial localization, homodimerization, as well as regulation of their pro-apoptotic activities (23Chen G. Ray R. Dubik D. Shi L. Cizeau J. Bleackley R.C. Saxena S. Gietz R.D. Greenberg A.H. J. Exp. Med. 1997; 186: 1975-1983Google Scholar). BNIP3-mediated apoptosis has been reported to be independent of caspase activation and cytochrome c release and is characterized by early plasma membrane and mitochondrial damage, prior to the appearance of chromatin condensation or DNA fragmentation (24Van de Velde C. Cizeau J. Dubik D. Alimonti J. Brown T. Israels S. Hakem R. Greenberg A.H. Mol. Cell. Biol. 2000; 20: 5454-5468Google Scholar). In the present work, we describe an original mechanism by which the BH3-only protein BNIP3 could be an important mediator of CD47-induced cell death. This pathway would be likely to act at sites where large amounts of soluble TSP-1 are available and could play an important role in T cell death in the vicinity of thrombotic events. Reagents—The CD47 mAb Ad22 was kindly provided by Dr. Rolf D. Pettersen (Departement of Pediatric Research and Pediatrics, The National Hospital, Oslo, Norway) and has been described elsewhere (9Pettersen R.D. Hestdal K. Olafsen M.K. Lie S.O. Lindberg F.P. J. Immunol. 1999; 162: 7031-7040Google Scholar). CD47 mAb B6H12 was from the American Type Culture Collection (ATCC, Rockville, MD). Rabbit polyclonal anti-BNIP3 and mouse monoclonal anti-BNIP3 Ana40 antibodies have been described elsewhere (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar). MAb against Fas CH11 was from Immunotech (BD Biosciences), mAb against Bcl-2 was from Santa Cruz Biotechnology, and mAb against V5 was from Invitrogen. SIRPα1-Fc fusion proteins have previously been described (25Rebres R.A. Green J.M. Reinhold M.I. Ticchioni M. Brown E.J. J. Biol. Chem. 2001; 276: 7672-7680Google Scholar). The peptides 4N1K (KRFYVVMWKK) and 4NGG (KRFYGGMWWKK) were from Genosys Biotechnologies (The Woodlands, TX). Plasmids encoding green fluorescent protein (GFP)-tagged cytochrome c and Bcl-2 (pCMV-Bcl-2) were kindly provided by Dr. A. Galmiche (INSERM U462, Nice, France). Cells—The Jurkat T cell line (JE6.1) was obtained from ATCC and cultured in RPMI 1640 (Invitrogen) supplemented with 5% fetal bovine serum (Dutcher, Brumath, France), 50 units/ml penicillin, 50 μg/ml streptomycin, 2 mm l-glutamine, and 1 mm pyruvate (Merck, Darmstadt, Germany). The CD47-deficient Jurkat T cell line (JIN for Jurkat IAP-negative cells) has been previously described (26Ticchioni M. Raimondi V. Lamy L. Wijdenes J. Lindberg F.P. Brown E.J. Bernard A. FASEB J. 2001; 15: 341-350Google Scholar). Assay for Apoptosis—Phosphatidylserine exposure and decrease in mitochondrial membrane potential (Δψm) were measured by flow cytometry using a FACScan (BD Biosciences). For phosphatidylserine exposure, cells were double-stained with annexin V-FITC and propidium iodide as described by the manufacturer (Roche Applied Science). The decrease in Δψm was assessed by incubating Jurkat cells with 40 nm 3,3′-dihexyloxacarbocyanine iodide (DiOC6) (Molecular Probes, Eugene, OR) for 30 min at 37 °C. Confocal Microscopy—Jurkat cells, treated or not with the pro-apoptotic 4N1K peptide for 2 h, were plated on polylysine glass slides (Menzel-Glaser, Freiburg, Germany). For mitochondrial staining, Texas Red MitoTracker (Molecular Probes) was added to the medium to a final concentration of 300 nm for 45 min. Cells were then fixed with 3.7% formaldehyde, permeabilized with 0.2% Triton X-100, and blocked for 20 min with phosphate-buffered saline containing bovine serum albumin (1%) and saponine (0.05%). Cells were stained for CD47 using SIRPα1-Fc and FITC-conjugated mouse anti-human antibody (Dako) at 4 °C before fixation, and for BNIP3 using the mouse monoclonal anti-BNIP3 antibody (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar) followed by a Cyan V-conjugated rabbit antimouse antibody. Samples were mounted in Mowiol (Calbiochem) and observed with a confocal microscope (Leica TCS-SP, Heidelberg, Germany). Co-immunoprecipitation and Western Blot Analysis—Cells were washed twice in cold phosphate-buffered saline and lysed in 2% Triton X-100 isotonic buffer with freshly added protease inhibitors (50 mm HEPES, pH 7.6, 150 mm NaCl, 20 mm EDTA, 10 mm sodium orthovana-date, 100 mm NaF, 2% Triton X-100, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 0.5 mm phenylmethylsulfonyl fluoride). Cell debris was removed by centrifugation and supernatants were incubated for 2 h at 4 °C with anti-CD47 antibody Ad22. Immunoprecipitation was performed with 40 μl protein G-Sepharose (Amersham Biosciences), for 4 h at 4 °C. The samples were washed three times in lysis buffer (0.5% Triton X-100), and immunoprecipitated proteins were analyzed on 10% SDS-polyacrylamide gel electrophoresis and immunoblotted with rabbit polyclonal anti-BNIP3 (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar) or anti-CD47 B6H12 antibodies. The immune complexes were detected by horseradish peroxydase-conjugated secondary antibodies (Dako) and developed using enhanced chemiluminescence (Amersham Biosciences). Cytochrome c Release—Cytosolic fractions were prepared by resuspending Jurkat cells (5 × 106) in 100 μl mitochondrial isolation buffer (MIB; 250 mm sucrose, 20 mm HEPES, 10 mm KCl, 2 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, and protease inhibitors). Cells were broken and homogenized by 10 passages through an ice-cold 26-gauge needle. Unlysed cells and nuclei were removed by centrifugation (5 min, 750 × g). The supernatant was spun at 10,000 × g for 30 min at 4 °C, and the resulting supernatant was saved as the cytosolic extract. Cytosolic extracts were separated on 15% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Amersham Biosciences). Blots were probed with mouse anti-cytochrome c antibody (65985A) (BD Bio-sciences) and anti-actin (Chemicon, Temecula, CA), incubated with horseradish peroxydase-conjugated secondary antibody (Dako), and developed using enhanced chemiluminescence (Amersham Biosciences). In a separate set of experiments, Jurkat cells were transfected with GFP-tagged cytochrome c, stimulated via CD47 or Fas 48 h after transfection, then labeled with Annexin V-PE as described by the manufacturer (Roche Applied Science). Cells were observed with a confocal microscope. Subcellular Fractionation—Jurkat cells (1 × 108) were stimulated with the mAb against CD47 Ad22 (1 μg/ml) for1hat37 °C or incubated in medium alone, washed once, and resuspended in chilled isotonic buffer (250 mm sucrose, 20 mm HEPES, 20 mm KCl, 5 mm MgCl2, 1 mm dithiothreitol, and protease inhibitors). Cells were broken using a Dounce homogeneizer (80 strokes with a tight-fitting pestle). The cell homogenate was first centrifuged at 1000 × g for 10 min. The post-nuclear supernatant was then centrifuged at 10,000 × g for 30 min. The pellet containing the mitochondrial fraction was resuspended in homogeneization buffer and further purified on a discontinuous metrizamide gradient (27Storrie B. Madden E.A. Methods Enzymol. 1990; 182: 203-225Google Scholar). The microsomal fraction contained in the 10,000 × g supernatant was collected by centrifugation (200,000 × g, 2 h). Cell extracts were analyzed by Western blotting using rabbit polyclonal anti-BNIP3 antibody, anti-translocase, a marker of the outer mitochondrial membrane (40 kDa; TOM40) (Interchim), and anti-placental alkaline phosphatase (Dako). Determination of Caspase 3 Activity—Each assay was performed in quadruplicate with 50 μg of protein as described previously (28Ricci J.E. Maulon L. Battaglione-Hofman V. Bertolotto C. Luciano F. Mari B. Hofman P. Auberger P. Eur. Cytokine Netw. 2001; 12: 126-134Google Scholar). Briefly, 4 × 106 cells were lysed in 0.2% Triton X-100 isotonic buffer (50 mm HEPES, pH 7.6, 150 mm NaCl, 20 mm EDTA) with freshly added protease inhibitors. Cellular extracts were then incubated in a 96-well plate with 10 mm dithiothreitol and 200 μm of Ac-DEVD-pNA as substrate for various times at 37 °C. Caspase activity was measured by the release of pNA at 410 nm in either the presence or absence of 1 μm Ac-DEVD-CHO, an irreversible inhibitor of caspases. The specific caspase activity represents the Ac-DEVD-CHO inhibitable activity and is expressed as nanomoles of substrate hydrolyzed per min and per mg of protein. BNIP3 Antisense Oligodeoxynucleotides—Two μm of BNIP3 anti-sense (sequence number 2.04252) or scrambled phosphorothioate oligodeoxynucleotides (Biognostik, Göttingen, Germany) were added to the cells at the start of culture and every time the culture medium was renewed during 72 h. Yeast Two-hybrid System—The C-terminal 145 amino acids of type 2 CD47 corresponding to the multispan and cytoplasmic portion of the molecule were fused to the LexA DNA-binding domain in pLex-9 (pLex-CD47 multiply membrane spanning (MMS)) and used as a bait for screening a human cDNA library (29Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Google Scholar, 30Foucault I. Liu Y.C. Bernard A. Deckert M. J. Biol. Chem. 2002; 24: 24Google Scholar). Standard techniques were used for nucleic acid manipulations and preparation of DNA constructs. Primers used to generate the DNA fragments were 5′-GGAATTCGGTATTAAAACACTTAAATATAGATCCGGT-3′ (sense oligonucleotide, EcoRI site underlined), and 5′-CGGGATCCTCAGTTATTCCTAGGAGGTTG-3′ (antisense oligonucleotides, BamHI site underlined, mutation introduced in bold). A second round of screening was done using a LexA-lamin plasmid as a negative control. CD47 MMS domain and BNIP3 interactions were assayed for growth on the basis of histidine prototrophy on DOBA Ura–Trp–Leu–His– plates. Subsequent testing for β-galactosidase activity was performed. Deletion mutants for BNIP3 in pcDNA3 vector were previously described (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar): BNIP3ΔN (Δ1–49), BNIP3ΔBH3 (Δ104–119), BNIP3ΔTM2 (Δ164–194), and BNIP3ΔC (Δ184–194). cDNAs were cloned into the EcoRI/BamHI sites of the yeast two-hybrid expression vector pActII. CD47 Induces a Rapid Mitochondrial Dysfunction and Apoptosis—As previously described (9Pettersen R.D. Hestdal K. Olafsen M.K. Lie S.O. Lindberg F.P. J. Immunol. 1999; 162: 7031-7040Google Scholar, 10Mateo V. Lagneaux L. Bron D. Biron G. Armant M. Delespesse G. Sarfati M. Nat. Med. 1999; 5: 1277-1284Google Scholar), CD47 antibody Ad22 in a soluble form induces a fast cell death with an early phosphatidylserine exposure (Fig. 1A) and mitochondrial abnormalities. Indeed, using DiOC6 to measure Δψm following cell stimulation via CD47 (Fig. 1B), we observed Δψm drop within 1 h, indicating that the mitochondrial dysfunction occurred as early as cell death. CD47 stimulation was nearly as efficient as CD95 at suppressing Δψm, as measured after optimal times. Despite early mitochondrial dysfunction following CD47 stimulation, we observed only a late and low release of cytochrome c which was undetectable before 3 h, in marked contrast with the effects of CD95 stimulation (Fig. 1C). Therefore, cytochrome c release appears to be a secondary event. To further ensure that cytochrome c release occurs after cell death, we transfected Jurkat cells with GFP-tagged cytochrome c and observed its mitochondrial release after CD47 stimulation while simultaneously measuring Annexin binding (Fig. 1D). Quite clearly, mitochondrial release was a later event. No role for caspases could be demonstrated by attempts to inhibit PCD with the broad-spectrum caspase inhibitor z-VAD-fmk (result not shown). Moreover, no activation of caspase 3 could be detected while PCD occurred in the same experiment, in marked contrast with controls measuring the effect of a CD95 antibody (Fig. 1E). CD47 Interacts with the Pro-apoptotic Molecule BNIP3—To determine the apoptotic pathway triggered by CD47 stimulation, we searched for CD47 interacting molecule(s) in the yeast two-hybrid system. We used the CD47 MMS domain fused to Gal4 as a bait for screening a human lymphocyte cDNA library. We ascertained the requirement for the MMS domain of CD47 to induce apoptosis by using a chimeric form of CD47, composed of the extracellular domain of CD47 and the membrane and cytoplasmic domains of CD7 (CD47ec-CD7) (15Reinhold M.I. Lindberg F.P. Kersh G.J. Allen P.M. Brown E.J. J. Exp. Med. 1997; 185: 1-11Google Scholar). This chimera was unable to transmit an apoptotic signal when transfected in the CD47-deficient T-cell line which we previously generated (JIN cells) (26Ticchioni M. Raimondi V. Lamy L. Wijdenes J. Lindberg F.P. Brown E.J. Bernard A. FASEB J. 2001; 15: 341-350Google Scholar), (Fig. 2A). CD47ec-CD7 mutants and wild-type CD47 transfected cells showed comparable levels of Fas-induced apoptosis (result not shown). Note that the use of MMS domain as bait to perform the two-hybrid screen has already been successively performed (31Xu X. Shi Y. Wu X. Gambetti P. Sui D. Cui M.Z. J. Biol. Chem. 1999; 274: 32543-32546Google Scholar, 32Alberici A. Moratto D. Benussi L. Gasparini L. Ghidoni R. Gatta L.B. Finazzi D. Frisoni G.B. Trabucchi M. Growdon J.H. Nitsch R.M. Binetti G. J. Biol. Chem. 1999; 274: 30764-30769Google Scholar, 33Passer B.J. Pellegrini L. Vito P. Ganjei J.K. D'Adamio L. J. Biol. Chem. 1999; 274: 24007-24013Google Scholar, 34Imafuku I. Masaki T. Waragai M. Takeuchi S. Kawabata M. Hirai S. Ohno S. Nee L.E. Lippa C.F. Kanazawa I. Imagawa M. Okazawa H. J. Cell Biol. 1999; 147: 121-134Google Scholar, 35Xu X. Shi Y.C. Gao W. Mao G. Zhao G. Agrawal S. Chisolm G.M. Sui D. Cui M.Z. J. Biol. Chem. 2002; 277: 48913-48922Google Scholar, 36Hebert S.S. Godin C. Tomiyama T. Mori H. Levesque G. Biochem. Biophys. Res. Commun. 2003; 301: 119-126Google Scholar). In the present screen, five positive clones were selected of an estimated 5 × 106 colonies, for their ability to reconstitute a functional Gal4 transcription complex. Among them, we found a clone encoding the pro-apoptotic BH3-only family protein BNIP3 (Fig. 2B). To determine the domain of the BNIP3 molecule involved in binding to CD47, we tested a series of BNIP3 deletion mutants (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar) by using the two-hybrid system. Whereas full-length BNIP3 showed a strong interaction with CD47, BNIP3ΔTM2 was unable to interact with CD47 (Fig. 2B) indicating that the transmembrane domain of BNIP3 is required for interaction with CD47. Mutants lacking the NH2 terminus, the BH3 domain, or the COOH terminus did not bind strongly to CD47 (Fig. 2B), suggesting that other regions in the full-length structure of BNIP3 are necessary for the tight interaction with CD47. To confirm the association between CD47 and BNIP3 in vivo, we performed co-immunoprecipitation assays (Fig. 2C). BNIP3 was specifically co-immunoprecipitated with CD47 from Jurkat T cells. Nevertheless, as expected, no co-immunoprecipitation was observed from JIN cells or cells expressing the CD47ec-CD7 mutant. Thus, BNIP3 interacts with the MMS domain of CD47 both in yeast and mammalian cells. Next, we investigated whether, by blocking BNIP3 synthesis with antisense oligodeoxynucleotides, the CD47-generated apoptotic signal could be interrupted. Jurkat cells were incubated with phosphorothioate-derivatized antisense oligodeoxynucleotides, and apoptosis in response to soluble CD47 antibody Ad22 or anti-Fas antibody CH11 was measured (Fig. 3A). Antisense oligodeoxynucleotides corresponding to BNIP3 blocked CD47-induced cell death, showing a 2-fold reduction in cell death, whereas a scrambled sequence of the same oligodeoxynucleotide had no effect. Moreover, oligodeoxynucleotides had no effect on either survival of control cells or on apoptosis induced by the anti-Fas antibody. Immunoblot analysis confirmed a loss of BNIP3 expression from cells cultured with the BNIP3 antisense oligodeoxynucleotides (Fig. 3A). Thus, reduction of BNIP3 expression specifically leads to a reduction of cell death induced via CD47. Finally, because it has been previously shown that BNIP3 binds Bcl-2 at the mitochondrial membrane, an event likely to play a role in BNIP3-dependent PCD (24Van de Velde C. Cizeau J. Dubik D. Alimonti J. Brown T. Israels S. Hakem R. Greenberg A.H. Mol. Cell. Biol. 2000; 20: 5454-5468Google Scholar), we induced overexpression of Bcl-2 in Jurkat cells. Over-expressing cells were less susceptible to PCD induced either via CD47 than their normal counterparts (Fig. 3B). Thus, reduction of BNIP3 expression or overexpression of Bcl-2 leads to a reduction of cell death induced via CD47, demonstrating a functional interaction between CD47 and BNIP3. TSP-1 Promotes CD47-induced Apoptosis and BNIP3 Translocation to Mitochondria—CD47 acts as a receptor both for the C-terminal domain of TSP-1 and for SIRPα1 (37Gao A.G. Lindberg F.P. Finn M.B. Blystone S.D. Brown E.J. Frazier W.A. J. Biol. Chem. 1996; 271: 21-24Google Scholar, 38Jiang P. Lagenaur C.F. Narayanan V. J. Biol. Chem. 1999; 274: 559-562Google Scholar). TSP-1 is a protein expressed on endothelial cells, found in large amounts in extracellular matrix and released by activated platelets, whereas SIRPα1 is expressed on the surface of macrophages and endothelial and dendritic cells. To determine which natural ligand induces cell death, we treated Jurkat cells or JIN cells with the CD47-binding agonist peptide from TSP-1 named 4N1K (37Gao A.G. Lindberg F.P. Finn M.B. Blystone S.D. Brown E.J. Frazier W.A. J. Biol. Chem. 1996; 271: 21-24Google Scholar) or with a recombinant SIRPα1-Fc fusion protein (25Rebres R.A. Green J.M. Reinhold M.I. Ticchioni M. Brown E.J. J. Biol. Chem. 2001; 276: 7672-7680Google Scholar) (Fig. 4A). At high concentrations (400 μm), soluble 4N1K induced a rapid death of Jurkat T cells, but had no nonspecific cytotoxic effect on CD47-deficient cells. Control peptide 4NGG failed to induce cell death in the two cell types. By contrast, the soluble SIRPα1-Fc fusion protein, which efficiently bound CD47 (results not shown), had no apoptotic effect, regardless of the amount of protein or the incubation time we used. It is worth noting that only high concentrations of 4N1K (400 μm) are able to induce cell death, whereas lower concentrations have been reported to sustain clonal expansion of inflammatory T cells (39Vallejo A.N. Mugge L.O. Klimiuk P.A. Weyand C.M. Goronzy J.J. J. Immunol. 2000; 164: 2947-2954Google Scholar) or to induce anergy of naive T cells (40Avice M.N. Rubio M. Sergerie M. Delespesse G. Sarfati M. J. Immunol. 2000; 165: 4624-4631Google Scholar, 41Li Z. He L. Wilson K. Roberts D. J. Immunol. 2001; 166: 2427-2436Google Scholar). This apoptotic effect of 4N1K on T cells is not due to a nonspecific cytotoxicity, because CD47-deficient cells are resistant to cell death induced by 4N1K. BNIP3 has been described to localize at and to act on the mitochondria when overexpressed (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar, 23Chen G. Ray R. Dubik D. Shi L. Cizeau J. Bleackley R.C. Saxena S. Gietz R.D. Greenberg A.H. J. Exp. Med. 1997; 186: 1975-1983Google Scholar, 42Boyd J.M. Malstrom S. Subramanian T. Venkatesh L.K. Schaeper U. Elangovan B. D'Sa-Eipper C. Chinnadurai G. Cell. 1994; 79: 341-351Google Scholar). The finding of a potential association between the cell surface CD47 molecule and BNIP3 prompted us to examine the subcellular localization of native BNIP3. We detected BNIP3 at the inner leaflet of the plasma membrane in addition to mitochondrial localization (results not shown). Because it was shown that BNIP3 is active when integrated in the outer membrane of the mitochondria, we have investigated whether the plasma membrane-associated BNIP3 molecule would relocalize to the mitochondria. To do so, we performed subcellular fractionations by ultracentrifugation of Jurkat cells stimulated with the CD47 antibody Ad22 or incubated in medium alone (Fig. 4B). In control cells incubated in medium alone, BNIP3 was detected both in the mitochondrial fraction, as assessed by the presence of TOM40, a protein localized on the outer mitochondrial membrane, and in the microsomal fraction including the plasma membrane, as assessed by the presence of placental alkaline phosphatase. Note that it is not possible to directly compare the microsomal and the mitochondrial fractions because the dilution factors of these fractions cannot be adjusted. When Jurkat cells were stimulated by the death-activating antibody, BNIP3 was no longer detected in the microsomal fraction, whereas its presence in the mitochondrial fraction appeared to be increased. To confirm that BNIP3 translocates from the plasma membrane to the mitochondria upon CD47 stimulation, we performed double labeling experiments in which T cells were stimulated or not with the pro-apoptotic 4N1K peptide and then double labeled for either CD47 or BNIP3, and mitochondria were revealed with the MitoTracker (Fig. 4C). When T cells were pretreated with the pro-apoptotic 4N1K peptide, BNIP3 was seen to localized to mitochondria, whereas substantial amounts of BNIP3 remained localized at the cell membrane in basal conditions (Fig. 4C). Our data demonstrate that a BH3-only protein can be pivotal in transducing an external signal via a surface receptor. So far BH3-only proteins have been mainly regarded as sensors of intracellular or stress-induced damages, and BNIP3 was shown to mediate hypoxia-induced apoptosis (43Bruick R.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9082-9087Google Scholar). However, the latter process appears to be slow, likely due to a requirement for de novo synthesis of BNIP3, because the molecule is quickly degraded by the proteasome (44Chen G. Cizeau J. Vande Velde C. Park J.H. Bozek G. Bolton J. Shi L. Dubik D. Greenberg A. J. Biol. Chem. 1999; 274: 7-10Google Scholar, 45Cizeau J. Ray R. Chen G. Gietz R.D. Greenberg A.H. Oncogene. 2000; 19: 5453-5463Google Scholar). It has been previously observed (21Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Google Scholar, 42Boyd J.M. Malstrom S. Subramanian T. Venkatesh L.K. Schaeper U. Elangovan B. D'Sa-Eipper C. Chinnadurai G. Cell. 1994; 79: 341-351Google Scholar) that, when overexpressed, BNIP3 localizes to mitochondria. Although we made the same observation, we also collected several lines of evidence demonstrating that under basal conditions a significant amount of BNIP3 is localized at the plasma membrane where it might associate with CD47. In a yeast two-hybrid system using the membrane-spanning domain of CD47 as bait, we identified BNIP3. More-over, immunoprecipitation of CD47 was accompanied by co-precipitation of BNIP3. Finally, double immunofluorescence visualization of CD47 and BNIP3 and subcellular fractionation revealed that a significant proportion of both molecules colocalize. The use of deleted forms of BNIP3 allowed us to identify the transmembrane domain of BNIP3 as the region necessary for interaction with the TM domains of CD47. A second series of experiments showed that BNIP3 is required for the pro-apoptotic effect of CD47. First, we observed a substantial reduction in CD47 pro-apoptotic effect when BNIP3 expression was markedly reduced, using an antisense oligodeoxynucleotide of BNIP3, or when the anti-apoptotic molecule Bcl-2, another BNIP3 interactor, was overexpressed. Because it has been shown that BNIP3 exerts its pro-apoptotic effect at the mitochondria membrane (22Yasuda M. Theodorakis P. Subramanian T. Chinnadurai G. J. Biol. Chem. 1998; 273: 12415-12421Google Scholar, 23Chen G. Ray R. Dubik D. Shi L. Cizeau J. Bleackley R.C. Saxena S. Gietz R.D. Greenberg A.H. J. Exp. Med. 1997; 186: 1975-1983Google Scholar, 44Chen G. Cizeau J. Vande Velde C. Park J.H. Bozek G. Bolton J. Shi L. Dubik D. Greenberg A. J. Biol. Chem. 1999; 274: 7-10Google Scholar), this suggests a simple model based on the translocation of BNIP3 from the inner surface of the cell membrane to the mitochondria. Consistent with this model, we showed that, after a pro-apoptotic signal transmitted via CD47 upon binding of a mAb or a peptide mimicking TSP-1, BNIP3 translocates from the plasma membrane to the mitochondria as assessed both by subcellular fractionation and immunofluorescence studies. This model accounts for a fast apoptosis and is consistent with the “rheostatic” effect exerted by the Bcl-2 family proteins (46Letai A. Bassik M. Walensky L. Sorcinelli M. Weiler S. Korsmeyer S. Cancer Cell. 2002; 2: 183Google Scholar, 47Cheng E.H. Wei M.C. Weiler S. Flavell R.A. Mak T.W. Lindsten T. Korsmeyer S.J. Mol. Cell. 2001; 8: 705-711Google Scholar, 48Adams J.M. Cory S. Science. 1998; 281: 1322-1326Google Scholar, 49Zimmermann K.C. Bonzon C. Green D.R. Pharmacol. Ther. 2001; 92: 57-70Google Scholar). It is worth noting that several other BH3-only proteins were reported to also be subjected to fast post-translational modification and translocation based effect (50Huang D.C. Strasser A. Cell. 2000; 103: 839-842Google Scholar). It must be emphasized, however, that BNIP3, together with Nix, forms a peculiar and quite conserved subfamily (45Cizeau J. Ray R. Chen G. Gietz R.D. Greenberg A.H. Oncogene. 2000; 19: 5453-5463Google Scholar, 51Yasuda M. D'Sa-Eipper C. Gong X.L. Chinnadurai G. Oncogene. 1998; 17: 2525-2530Google Scholar) within the BH3-only proteins. Their BH3 domain was found to be non-functional in terms of association with the anti-apoptotic proteins Bcl-2 and Bcl-XL. Rather it was demonstrated that their transmembrane C-terminal segment as well as N-terminal residues are necessary for these associations. Although a series of observations have shown that “classical” BH3-only protein associates with BAX and BAK to induce cell death (46Letai A. Bassik M. Walensky L. Sorcinelli M. Weiler S. Korsmeyer S. Cancer Cell. 2002; 2: 183Google Scholar, 52Martinou J.C. Green D.R. Nat. Rev. Mol. Cell. Biol. 2001; 2: 63-67Google Scholar), the mitochondrial and post-mitochondrial mechanisms leading to cell death with the BNIP3 subfamily remain to be established. Moreover, whether other BH3-only proteins would mediate apoptotic signals transduced via distinct transmembrane receptors remains to be determined. T cell PCD via CD47 can be triggered by TSP-1 and not by its other natural ligand SIRPα1. The fact that only high amounts of TSP-1 can trigger apoptosis also fits with the above model. By contrast, lower amounts of TSP-1 and 4N1K can sustain clonal expansion of T cells (14Ticchioni M. Deckert M. Mary F. Bernard G. Brown E.J. Bernard A. J. Immunol. 1997; 158: 677-684Google Scholar, 15Reinhold M.I. Lindberg F.P. Kersh G.J. Allen P.M. Brown E.J. J. Exp. Med. 1997; 185: 1-11Google Scholar, 16Waclavicek M. Majdic O. Stulnig T. Berger M. Baumruker T. Knapp W. Pickl W.F. J. Immunol. 1997; 159: 5345-5354Google Scholar). It can therefore be assumed that this pro-apoptotic pathway could quickly limit the inflammatory response, which tends to develop after thrombosis. We have collected in vivo evidence, in an inflammation model, for such a role of CD47. 2L. Lamy, M. Ticchioni, A. Foussat, E. J. Brown, and A. Bernard, manuscript in preparation. Consistent with this view, it must be kept in mind that the CD47 pro-apoptotic pathway acts only on activated, but not resting, normal T cells (9Pettersen R.D. Hestdal K. Olafsen M.K. Lie S.O. Lindberg F.P. J. Immunol. 1999; 162: 7031-7040Google Scholar). Interestingly, an overexpression of TSP-1 has recently been observed after myocardial infarction, forming a belt around injured tissues (55Ren G. Mendoza L.H. Jackson P. Michael L.H. Smith W.C. Entman M.L. Frangogiannis N.G. FASEB J. 2002; 16 (Abstr.): A541Google Scholar). Moreover, cell types other than T cells could be subjected to an apoptotic regulation via the TSP-1/CD47 pathway, as both TSP-1 and CD47 have been reported to be strongly up-regulated on vascular endothelial cells upon disturbance of blood laminar flow and can induce their apoptosis (53Freyberg M.A. Kaiser D. Graf R. Vischer P. Friedl P. Biochem. Biophys. Res. Commun. 2000; 271: 584-588Google Scholar, 54Freyberg M.A. Kaiser D. Graf R. Buttenbender J. Friedl P. Biochem. Biophys. Res. Commun. 2001; 286: 141-149Google Scholar). We thank E. van Obberghen-Schilling and K. E. Boulukos for critical reading of the manuscript, and we thank members of the laboratory, especially I. Foucault, for discussions. We are grateful to R. D. Pettersen for kindly providing Ad22 mAb, R. A. Rebres for SIRPα1-Fc fusion protein, D. Dubik for antibodies against BNIP3 and for deletion mutants for BNIP3, A. Galmiche for Bcl-2 and cytochrome c-GFP constructs, and P. Auberger for caspase measurements." @default.
• W2019905662 created "2016-06-24" @default.
• W2019905662 creator A5003744102 @default.
• W2019905662 creator A5005517169 @default.
• W2019905662 creator A5007553713 @default.
• W2019905662 creator A5021986215 @default.
• W2019905662 creator A5045375697 @default.
• W2019905662 creator A5055348027 @default.
• W2019905662 creator A5081330050 @default.
• W2019905662 date "2003-06-01" @default.
• W2019905662 modified "2023-10-14" @default.
• W2019905662 title "CD47 and the 19 kDa Interacting Protein-3 (BNIP3) in T Cell Apoptosis" @default.
• W2019905662 cites W146759131 @default.
• W2019905662 cites W1530982587 @default.
• W2019905662 cites W1554372630 @default.
• W2019905662 cites W1592843561 @default.
• W2019905662 cites W1933742170 @default.
• W2019905662 cites W1964617786 @default.
• W2019905662 cites W1976720365 @default.
• W2019905662 cites W1980024819 @default.
• W2019905662 cites W1991043791 @default.
• W2019905662 cites W1998225655 @default.
• W2019905662 cites W2000923801 @default.
• W2019905662 cites W2002131156 @default.
• W2019905662 cites W2003319634 @default.
• W2019905662 cites W2005528779 @default.
• W2019905662 cites W2017555329 @default.
• W2019905662 cites W2019784233 @default.
• W2019905662 cites W2026225494 @default.
• W2019905662 cites W2028409421 @default.
• W2019905662 cites W2028890259 @default.
• W2019905662 cites W2033935793 @default.
• W2019905662 cites W2034796667 @default.
• W2019905662 cites W2044441834 @default.
• W2019905662 cites W2054385312 @default.
• W2019905662 cites W2056418403 @default.
• W2019905662 cites W2060480804 @default.
• W2019905662 cites W2061475369 @default.
• W2019905662 cites W2061822552 @default.
• W2019905662 cites W2064967819 @default.
• W2019905662 cites W2066829203 @default.
• W2019905662 cites W2082195780 @default.
• W2019905662 cites W2083043223 @default.
• W2019905662 cites W2083359402 @default.
• W2019905662 cites W2093455126 @default.
• W2019905662 cites W2096916633 @default.
• W2019905662 cites W2100577875 @default.
• W2019905662 cites W2119228169 @default.
• W2019905662 cites W2119800239 @default.
• W2019905662 cites W2124784466 @default.
• W2019905662 cites W2131484344 @default.
• W2019905662 cites W2144243712 @default.
• W2019905662 cites W2150087633 @default.
• W2019905662 cites W2152247516 @default.
• W2019905662 cites W2168324886 @default.
• W2019905662 cites W2318392474 @default.
• W2019905662 cites W2324579067 @default.
• W2019905662 doi "https://doi.org/10.1074/jbc.m301869200" @default.
• W2019905662 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12690108" @default.
• W2019905662 hasPublicationYear "2003" @default.
• W2019905662 type Work @default.
• W2019905662 sameAs 2019905662 @default.
• W2019905662 citedByCount "82" @default.
• W2019905662 countsByYear W20199056622012 @default.
• W2019905662 countsByYear W20199056622013 @default.
• W2019905662 countsByYear W20199056622014 @default.
• W2019905662 countsByYear W20199056622015 @default.
• W2019905662 countsByYear W20199056622016 @default.
• W2019905662 countsByYear W20199056622017 @default.
• W2019905662 countsByYear W20199056622018 @default.
• W2019905662 countsByYear W20199056622019 @default.
• W2019905662 countsByYear W20199056622020 @default.
• W2019905662 countsByYear W20199056622021 @default.
• W2019905662 countsByYear W20199056622022 @default.
• W2019905662 countsByYear W20199056622023 @default.
• W2019905662 crossrefType "journal-article" @default.
• W2019905662 hasAuthorship W2019905662A5003744102 @default.
• W2019905662 hasAuthorship W2019905662A5005517169 @default.
• W2019905662 hasAuthorship W2019905662A5007553713 @default.
• W2019905662 hasAuthorship W2019905662A5021986215 @default.
• W2019905662 hasAuthorship W2019905662A5045375697 @default.
• W2019905662 hasAuthorship W2019905662A5055348027 @default.
• W2019905662 hasAuthorship W2019905662A5081330050 @default.
• W2019905662 hasBestOaLocation W20199056621 @default.
• W2019905662 hasConcept C1491633281 @default.
• W2019905662 hasConcept C153911025 @default.
• W2019905662 hasConcept C185592680 @default.
• W2019905662 hasConcept C190283241 @default.
• W2019905662 hasConcept C2780104668 @default.
• W2019905662 hasConcept C31573885 @default.
• W2019905662 hasConcept C55493867 @default.
• W2019905662 hasConcept C86803240 @default.
• W2019905662 hasConcept C95444343 @default.
• W2019905662 hasConceptScore W2019905662C1491633281 @default.
• W2019905662 hasConceptScore W2019905662C153911025 @default.
• W2019905662 hasConceptScore W2019905662C185592680 @default.
• W2019905662 hasConceptScore W2019905662C190283241 @default.
• W2019905662 hasConceptScore W2019905662C2780104668 @default.

• next