Neurotrophin Receptor Interacting Factor (NRIF) Is an Essential Mediator of Apoptotic Signaling by the p75 Neurotrophin Receptor
2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês
10.1074/jbc.m410435200
ISSN1083-351X
AutoresMichelle S. Linggi, Tara L. Burke, B. Blairanne Williams, Anthony Harrington, Rosemary Kraemer, Barbara L. Hempstead, Sung Ok Yoon, Bruce Carter,
Tópico(s)Nuclear Receptors and Signaling
ResumoActivation of the p75 neurotrophin receptor leads to a variety of effects within the nervous system, including neuronal apoptosis. Both c-Jun N-terminal kinase (JNK) and the tumor suppressor p53 have been reported to be critical for this receptor to induce cell death; however, the mechanisms by which p75 activates these pathways is undetermined. Here we report that the neurotrophin receptor interacting factor (NRIF) is necessary for p75-dependent JNK activation and apoptosis. Upon nerve growth factor withdrawal, nrif–/– sympathetic neurons underwent apoptosis, whereas p75-mediated death was completely abrogated. The lack of cell death correlated with a lack of JNK activation in the nrif–/– neurons, suggesting that NRIF is a selective mediator for p75-dependent JNK activation and apoptosis. Moreover, we document that NRIF expression is sufficient to induce cell death through a mechanism that requires p53. Taken together, these results establish NRIF as an essential component of the p75 apoptotic pathway. Activation of the p75 neurotrophin receptor leads to a variety of effects within the nervous system, including neuronal apoptosis. Both c-Jun N-terminal kinase (JNK) and the tumor suppressor p53 have been reported to be critical for this receptor to induce cell death; however, the mechanisms by which p75 activates these pathways is undetermined. Here we report that the neurotrophin receptor interacting factor (NRIF) is necessary for p75-dependent JNK activation and apoptosis. Upon nerve growth factor withdrawal, nrif–/– sympathetic neurons underwent apoptosis, whereas p75-mediated death was completely abrogated. The lack of cell death correlated with a lack of JNK activation in the nrif–/– neurons, suggesting that NRIF is a selective mediator for p75-dependent JNK activation and apoptosis. Moreover, we document that NRIF expression is sufficient to induce cell death through a mechanism that requires p53. Taken together, these results establish NRIF as an essential component of the p75 apoptotic pathway. The p75 neurotrophin receptor is a pleiotropic signaling molecule that regulates cellular survival, neurite outgrowth, and myelin formation (1.Barker P.A. Neuron. 2004; 42: 529-533Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). This founding member of the tumor necrosis factor receptor superfamily can directly bind all of the neurotrophins, including nerve growth factor (NGF), 1The abbreviations used are: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; JNK, c-Jun N-terminal kinase; GFP, green fluorescent protein; MEF, mouse embryo fibroblast; ProNGF, proform of NGF; DAPI, 4′,6-diamidino-2-phenylindole; NRIF, neurotrophin receptor interacting factor; MOPS, 4-morpholinepropanesulfonic acid; TRAF2, tumor necrosis factor receptor-associated factor 2; TrkA, tropomylosin-related kinase A. brain-derived neurotrophic factor (BDNF), and neurotrophin-3 and -4 (NT-3, NT-4), but it also functions as a co-receptor in a variety of protein complexes. p75 can interact with TrkA to form a high affinity neurotrophin binding site (2.Hempstead B.L. Martin-Zanca D. Kaplan D.R. Parada L.F. Chao M.V. Nature. 1991; 350: 678-683Crossref PubMed Scopus (1022) Google Scholar) and enhance survival signaling (3.Barker P.A. Shooter E.M. Neuron. 1994; 13: 203-215Abstract Full Text PDF PubMed Scopus (369) Google Scholar). It can also associate with the Nogo receptor and Lingo-1 and, upon interaction with myelin proteins, block neurite outgrowth (4.Wang K.C. Kim J.A. Sivasankaran R. Segal R. He Z. Nature. 2002; 420: 74-78Crossref PubMed Scopus (719) Google Scholar, 5.Mi S. Lee X. Shao Z. Thill G. Ji B. Relton J. Levesque M. Allaire N. Perrin S. Sands B. Crowell T. Cate R.L. McCoy J.M. Pepinsky R.B. Nat. Neurosci. 2004; 7: 221-228Crossref PubMed Scopus (709) Google Scholar), and, together with Sortilin, the neurotensin 3 receptor, it binds the proform of NGF and initiates apoptosis (6.Nykjaer A. Lee R. Teng K.K. Jansen P. Madsen P. Nielsen M.S. Jacobsen C. Kliemannel M. Schwarz E. Willnow T.E. Hempstead B.L. Petersen C.M. Nature. 2004; 427: 843-848Crossref PubMed Scopus (800) Google Scholar). The divergent cellular responses depend on the receptor complex as well as the cellular context. For example, sympathetic neurons of superior cervical ganglia undergo a period of programmed cell death during ontogenesis, and NGF, supplied by the tissues innervated, prevents the loss of these neurons through binding to a p75-TrkA complex (7.Chao M.V. Hempstead B.L. Trends Neurosci. 1995; 18: 321-326Abstract Full Text PDF PubMed Scopus (778) Google Scholar). In contrast, specific activation of p75 (8.Bamji S.X. Majdan M. Pozniak C.D. Belliveau D.J. Aloyz R. Kohn J. Causing C.G. Miller F.D. J. Cell Biol. 1998; 140: 911-923Crossref PubMed Scopus (443) Google Scholar) or a p75-sortilin complex (6.Nykjaer A. Lee R. Teng K.K. Jansen P. Madsen P. Nielsen M.S. Jacobsen C. Kliemannel M. Schwarz E. Willnow T.E. Hempstead B.L. Petersen C.M. Nature. 2004; 427: 843-848Crossref PubMed Scopus (800) Google Scholar) induces apoptosis in the neurons. Genetic deletion of p75 prevents the normal period of cell death in the developing superior cervical ganglia (8.Bamji S.X. Majdan M. Pozniak C.D. Belliveau D.J. Aloyz R. Kohn J. Causing C.G. Miller F.D. J. Cell Biol. 1998; 140: 911-923Crossref PubMed Scopus (443) Google Scholar, 9.Brennan C. Rivas-Plata K. Landis S.C. Nat. Neurosci. 1999; 2: 699-705Crossref PubMed Scopus (107) Google Scholar), thus demonstrating the key role of this receptor in regulating the survival of this neuronal population. How p75 initiates this variety of biological effects is not well understood; however, the stress kinase c-Jun N-terminal kinase, JNK, has been suggested to play a role in mediating this apoptotic signal. Neurotrophin activation of JNK through p75 correlates with the induction of cell death (43.Casaccia-Bonnefil P. Carter B.D. Dobrowsky R.T. Chao M.V. Nature. 1996; 383: 716-719Crossref PubMed Scopus (719) Google Scholar) and inhibition of the kinase prevents the receptor from killing (23.Yoon S.O. Casaccia-Bonnefil P. Carter B. Chao M.V. J. Neurosci. 1998; 18: 3273-3281Crossref PubMed Google Scholar, 10.Harrington A.W. Kim J.Y. Yoon S.O. J. Neurosci. 2002; 22: 156-166Crossref PubMed Google Scholar). Interestingly, c-Jun, the downstream target of the kinase, is not required for the receptor to activate apoptosis (11.Palmada M. Kanwal S. Rutkoski N.J. Gustafson-Brown C. Johnson R.S. Wisdom R. Carter B.D. J. Cell Biol. 2002; 158: 453-461Crossref PubMed Scopus (108) Google Scholar); however, other JNK substrates have been implicated in the p75 death pathway, including the pro-apoptotic Bcl-2 family member Bad (12.Bhakar A.L. Howell J.L. Paul C.E. Salehi A.H. Becker E.B. Said F. Bonni A. Barker P.A. J. Neurosci. 2003; 23: 11373-11381Crossref PubMed Google Scholar) and the tumor suppressor p53 (13.Aloyz R.S. Bamji S.X. Pozniak C.D. Toma J.G. Atwal J. Kaplan D.R. Miller F.D. J. Cell Biol. 1998; 143: 1691-1703Crossref PubMed Scopus (260) Google Scholar). p53 was suggested to function in p75 signaling, because expression of the viral p53 inhibitor E1B prevented the receptor from inducing apoptosis in sympathetic neurons and mice lacking p53 show a significant reduction in the normal attrition of sympathetic neurons during development (13.Aloyz R.S. Bamji S.X. Pozniak C.D. Toma J.G. Atwal J. Kaplan D.R. Miller F.D. J. Cell Biol. 1998; 143: 1691-1703Crossref PubMed Scopus (260) Google Scholar). A number of proteins have also been identified that can bind the intracellular domain of the receptor (1.Barker P.A. Neuron. 2004; 42: 529-533Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), and several have been implicated in regulating apoptosis, including NRAGE (14.Salehi A.H. Roux P.P. Kubu C.J. Zeindler C. Bhakar A. Tannis L.L. Verdi J.M. Barker P.A. Neuron. 2000; 27: 279-288Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), NADE (15.Mukai J. Hachiya T. Shoji-Hoshino S. Kimura M.T. Nadano D. Suvanto P. Hanaoka T. Li Y. Irie S. Greene L.A. Sato T.A. J. Biol. Chem. 2000; 275: 17566-17570Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) and NRIF (16.Casademunt E. Carter B.D. Benzel I. Frade J.M. Dechant G. Barde Y.A. EMBO J. 1999; 18: 6050-6061Crossref PubMed Scopus (156) Google Scholar); however, evidence demonstrating a direct requirement for any of these proteins in p75-mediated apoptosis is lacking. Moreover, the mechanisms through which they regulate cell death pathways remain largely unknown. In the present study, we examine the role of NRIF (neurotrophin receptor interacting factor, also known as ZFP 110) in p75-mediated cell death. NRIF was isolated in a yeast two-hybrid screen and encodes a 94-kDa zinc finger protein of the Krüppel family (16.Casademunt E. Carter B.D. Benzel I. Frade J.M. Dechant G. Barde Y.A. EMBO J. 1999; 18: 6050-6061Crossref PubMed Scopus (156) Google Scholar). It was suggested to have role in p75-mediated apoptosis based on the fact that mice lacking nrif display a significant reduction in cell death in the developing retina (16.Casademunt E. Carter B.D. Benzel I. Frade J.M. Dechant G. Barde Y.A. EMBO J. 1999; 18: 6050-6061Crossref PubMed Scopus (156) Google Scholar), a phenotype also observed in p75 null mice (17.Frade J.M. Rodriguez-Tebar A. Barde Y.A. Nature. 1996; 383: 166-168Crossref PubMed Scopus (668) Google Scholar). Despite this in vivo correlative evidence, it remains to be shown that NRIF is directly involved in signaling through the p75 receptor. Here, we demonstrate that NRIF is required for p75-mediated apoptosis of sympathetic neurons, but is dispensable for cell death after NGF withdrawal. In addition, we establish a link between NRIF and both of the known components of p75 apoptosis signaling, JNK and p53. Cell Culture—Mice lacking nrif were maintained on a 129Sv background and genotyped as previously described (16.Casademunt E. Carter B.D. Benzel I. Frade J.M. Dechant G. Barde Y.A. EMBO J. 1999; 18: 6050-6061Crossref PubMed Scopus (156) Google Scholar) and p53–/– mice on a mixed background were genotyped as previously described (18.Jacks T. Remington L. Williams B.O. Schmitt E.M. Halachmi S. Bronson R.T. Weinberg R.A. Curr. Biol. 1994; 4: 1-7Abstract Full Text Full Text PDF PubMed Scopus (1746) Google Scholar). Sympathetic neurons were isolated from the superior cervical ganglia as described by Palmada et al. (11.Palmada M. Kanwal S. Rutkoski N.J. Gustafson-Brown C. Johnson R.S. Wisdom R. Carter B.D. J. Cell Biol. 2002; 158: 453-461Crossref PubMed Scopus (108) Google Scholar). Briefly, superior cervical ganglia from nrif or p53 wild type, heterozygous, or null animals were isolated at postnatal days 2–4, and the sympathetic neurons were dissociated with 0.25% trypsin and 0.3% collagenase for 30 min at 37 °C. The nonneuronal cells were removed with a 2-h preplating on uncoated, Falcon 60-mm plates (BD Biosciences). The neurons were cultured on poly-l-ornithine and laminin-coated 4-well slides (Nalge Nunc International) in Ultraculture medium (BioWhittaker) supplemented with 3% fetal calf serum (Invitrogen), 2 mm l-glutamine (Invitrogen), and 20 ng/ml NGF (Harlan). The neurons were maintained for 4–5 days in the presence of NGF before being used for survival assays in NGF withdrawal and p75 activation experiments. Rat Schwann cells were isolated from postnatal day 4 rats as described by Carter et al. (19.Carter B.D. Kaltschmidt C. Kaltschmidt B. Offenhauser N. Bohm-Matthaei R. Baeuerle P.A. Barde Y.A. Science. 1996; 272: 542-545Crossref PubMed Scopus (614) Google Scholar) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2 μm forskolin (Sigma). Cells were transfected with Effectene (Qiagen) per the manufacturer's instructions. When adenovirally infected, cells were split the day before infection and infected with 4.5 × 106 plaque-forming units/cell of virus expressing NRIF and GFP bicistronically or GFP alone for mouse embryo fibroblasts or 7.5 × 106 plaque-forming units/cell for sympathetic neurons or Schwann cells. At the indicated times, cells were harvested for staining or immunoblotting, as indicated. Mouse embryo fibroblasts (MEFs) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cultures were split every 3 days, and all experiments were done with cells that had been passaged fewer than 12 times. ProNGF Production—ProNGF was generated by transfection of HEK 293 cells with a furin-resistant, His-tagged construct, and the protein was purified using nickel-bead chromatography (Xpress System Protein purification, Invitrogen) as per the manufacturer's instructions using imidazole for elution, as previously described (21.Lee R. Kermani P. Teng K.K. Hempstead B.L. Science. 2001; 294: 1945-1948Crossref PubMed Scopus (1390) Google Scholar). Mature NGF was similarly produced and used for comparison in all experiments with ProNGF. Survival Assays—For NGF withdrawal experiments, NGF was removed by washing the cultures twice in Ultraculture medium lacking NGF, and once with Ultraculture containing an antibody to NGF at 0.1 μg/ml (Chemicon International). The procedure was similar for the p75 activation experiments except that after the anti-NGF wash, the neurons were switched to media containing anti-NGF together with 12.5 mm KCl, to promote survival, with or without 200 ng/ml BDNF (a gift from Regeneron Pharmaceuticals, Inc.). Forty-eight hours after the switch to NGF-free or BDNF-containing media, the cells were fixed in 4% paraformaldehyde and the number of surviving neurons, identified by DAPI staining the nuclei (Vector Laboratories), were counted. The induction of cell death was confirmed by evaluating condensed or fragmented nuclei, which was also verified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining in some experiments. For proNGF treatment, the neurons were plated directly in media containing 4.6 mm imidazole (used to elute pro- or mature NGF off the nickel beads), 20 ng/ml NGF or proNGF and fixed 20 h later. Schwann cells and MEFs infected with adeno-NRIF or GFP were similarly scored for apoptosis. In the assays done with infected cells, only GFP-expressing cells were quantified. In all cases, at least 100 cells per condition were counted. NF-κB Activation Assays—Activation of NF-κB was assessed in primary cultures of Schwann cells, 1 or 2 days after isolation, using a luciferase reporter 6XκB-Luc (a gift from Larry Kerr). The cells were transfected with 0.2 μg of 6×κB-Luc reporter and 0.02 μg of Rous sarcoma virus-Renilla (used as internal control for transfection efficiency) per well of a 24-well plate using Effectene (Qiagen) according to the manufacturer's protocol. After 24 h, the cells were washed twice in serum-free Dulbecco's modified Eagle's medium and treated with 100 ng/ml NGF for 4–6 h and lysed in 40 μl of reporter lysis buffer (Promega). Luciferase activity was measured according to the manufacturer's instructions (Promega) using a luminometer (Monolight 2010, Analytical Luminescence Laboratory). The results were normalized to the basal activity for each treatment and genotype. There was no consistent difference in the basal activity between genotypes, although there was considerable variability. Immunostaining—Rat Schwann cells were maintained as described above. After transfection with adeno-GFP or GFP-NRIF, cells were fixed in 4% paraformaldehyde, blocked with 10% goat serum in PT (phosphate-buffered saline, 0.1% Triton X-100) and immunostained with antiserum to caspase 3 (a gift from Idun Pharmaceuticals, Inc.) diluted 1:1000 in PT, followed by a biotinylated secondary antibody (Vector Laboratories) and Cy-3 streptavidin. Nuclei were visualized by DAPI, and slides were viewed by fluorescence microscopy with a Zeiss Axiophot microscope. Sympathetic neurons were isolated and treated with BDNF as described. Twenty hours after treatment, neurons were fixed in 4% paraformaldehyde, permeabilized with 0.4% Triton X-100, blocked with 5% goat serum in PT, and immunostained with 1 μg/ml phospho-JNK antiserum (BIOSOURCE) overnight, followed by anti-rabbit Alexa 546-conjugated secondary antibody. For c-Jun immunostaining, cells were permeabilized with 0.1% sodium citrate and 0.1% Triton X-100, blocked in 10% goat serum, and immunostained with antiserum to c-Jun (Cell Signaling), diluted 1:500, followed by biotinylated secondary antibody (Vector Laboratories) and Cy-3 streptavidin. Nuclei were visualized by DAPI and viewed by fluorescence microscopy as described. Western Analysis—Schwann cells were cultured and transfected as described and 18 h later, mitochondrially enriched fractions were prepared as described (20.Lipscomb E.A. Sarmiere P.D. Freeman R.S. J. Biol. Chem. 2001; 276: 5085-5092Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Briefly, each plate was rinsed twice in cold phosphate-buffered saline before adding homogenization buffer (17 mm MOPS, 2.5 mm EDTA, 250 mm sucrose, 0.1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, and 5 μg/ml aprotinin). Cells were scraped from the dishes, Dounce-homogenized, and centrifuged at 750 × g for 10 min to pellet nuclei. The supernatants were centrifuged at 10,000 × g for 15 min, and the resultant pellets were resuspended in homogenization buffer and centrifuged again at 10,000 × g for 15 min to enrich for mitochondria. The mitochondrial fraction was suspended in RIPA buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% IGEPAL, 0.5% deoxycholate, 0.1% SDS, 0.5 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin) and analyzed by Western blotting using antibodies at the following dilutions: 1:1000 cytochrome c (BD Pharmingen) and 1:1000 manganese superoxide dismutase (BD Biosciences). Sympathetic neurons were isolated and treated with BDNF as described. Neurons from both CD-1 and 129Sv mice were used as control for comparison to those from nrif–/– animals; however, there was no difference between the control strains in the ability of BDNF to activate the kinase. Twenty-four hours after treatment, neurons were lysed in lysis buffer (2% SDS, 20% glycerol, 0.1 m Tris-HCl (pH 6.8) with protease inhibitors 0.5 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 2 μg/ml aprotinin) and analyzed by Western blotting using antibodies at the following dilutions: 1:500 JNK (Santa Cruz Biotechnology) and 1:500 phospho-JNK (Cell Signaling). MEFs were infected as described or treated with 60 J/mm2 ultraviolet light and 24 h later, cells were lysed in radioimmune precipitation assay buffer, and analyzed by Western blotting using JNK and phospho-JNK antibodies as above. NRIF Is Required for Apoptosis Mediated by the p75 Receptor—NRIF was first suggested to have a role in p75-mediated apoptosis based on the fact that mice lacking the nrif gene exhibit a reduction in the amount of naturally occurring cell death in the embryonic retina (16.Casademunt E. Carter B.D. Benzel I. Frade J.M. Dechant G. Barde Y.A. EMBO J. 1999; 18: 6050-6061Crossref PubMed Scopus (156) Google Scholar), a process known to depend on the p75 neurotrophin receptor (17.Frade J.M. Rodriguez-Tebar A. Barde Y.A. Nature. 1996; 383: 166-168Crossref PubMed Scopus (668) Google Scholar). Therefore, to directly investigate whether nrif is essential for p75-mediated apoptosis we activated the receptor in sympathetic neurons isolated from the superior cervical ganglia from nrif+/+, +/–, and –/– mice. Sympathetic neurons require NGF binding the tyrosine kinase receptor TrkA for survival; however, they can be maintained in the absence of neurotrophin using 12.5 mm KCl. These conditions allow selective activation of the p75 receptor by BDNF, resulting in neuronal apoptosis (8.Bamji S.X. Majdan M. Pozniak C.D. Belliveau D.J. Aloyz R. Kohn J. Causing C.G. Miller F.D. J. Cell Biol. 1998; 140: 911-923Crossref PubMed Scopus (443) Google Scholar, 11.Palmada M. Kanwal S. Rutkoski N.J. Gustafson-Brown C. Johnson R.S. Wisdom R. Carter B.D. J. Cell Biol. 2002; 158: 453-461Crossref PubMed Scopus (108) Google Scholar). Using this experimental paradigm, we found that neurons from the nrif null mice do not die in response to BNDF, in contrast to neurons from+/+ and +/– littermates (Fig. 1A). Recent data has suggested that the proform of NGF (proNGF) can also induce apoptosis in these neurons through interaction with the p75 receptor (21.Lee R. Kermani P. Teng K.K. Hempstead B.L. Science. 2001; 294: 1945-1948Crossref PubMed Scopus (1390) Google Scholar). Similar to the results with BDNF, the nrif–/– neurons were resistant to proNGF (Fig. 1B). These results indicate that NRIF is essential for p75 to signal neuronal apoptosis. The difference in the response to p75 activation between nrif–/– and +/+ was not due to loss of p75 expression (data not shown) nor was there any difference in general neuronal viability between the two genotypes. The NGF-mediated survival of the nrif–/– neurons was not different from the nrif+/+ cells (relative to wild type, 110 ± 29% of the nrif–/– neurons were viable after 48 h in the presence of 20 ng/ml NGF) nor was the survival in KCl (relative to wild type, 99 ± 16% of the nrif–/– neurons were viable). In addition, there was no difference in the degree of apoptosis in response to NGF withdrawal between the genotypes (Fig. 2). Thus, although NRIF is required for p75-mediated cell death, it is not required for apoptosis that occurs when neurons are deprived of NGF. The Activation of JNK by p75 Is Attenuated in the Absence of NRIF—Activation of the stress kinase JNK by p75 has been shown to be necessary for the receptor to induce cell death (10.Harrington A.W. Kim J.Y. Yoon S.O. J. Neurosci. 2002; 22: 156-166Crossref PubMed Google Scholar, 22.Friedman W.J. J. Neurosci. 2000; 20: 6340-6346Crossref PubMed Google Scholar, 23.Yoon S.O. Casaccia-Bonnefil P. Carter B. Chao M.V. J. Neurosci. 1998; 18: 3273-3281Crossref PubMed Google Scholar). The mechanism of JNK activation by p75 has not been delineated; however, TRAF6, a member of the tumor necrosis factor receptor associated factor family (25.Khursigara G. Bertin J. Yano H. Moffett H. DiStefano P.S. Chao M.V. J. Neurosci. 2001; 21: 5854-5863Crossref PubMed Google Scholar), and the GTP-binding protein Rac (10.Harrington A.W. Kim J.Y. Yoon S.O. J. Neurosci. 2002; 22: 156-166Crossref PubMed Google Scholar) have been implicated as upstream activators. Furthermore, it was recently reported that co-expression of NRIF, TRAF6, and p75 in 293 HEK cells reconstituted JNK activation by neurotrophin (24.Gentry J.J. Rutkoski N.J. Burke T.L. Carter B.D. J. Biol. Chem. 2004; 279: 16646-16656Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Therefore, we evaluated the stimulation of this kinase in neurons from mice lacking nrif. Sympathetic neurons isolated from nrif–/– or wild type mice were cultured in KCl, as for the cell death assays, treated with BDNF for 24 h and the activation of JNK assessed by Western blotting for phospho-JNK. Unlike wild type neurons, there was no JNK activation detectable in those from nrif–/– mice (Fig. 3A). The total level of JNK was not significantly different between the wild type and null neurons (based on normalization to tubulin and a Student's t test, p = 0.36, n = 4). In addition, we evaluated the stimulation of this kinase by immunostaining for phospho-JNK. Approximately 80% of the wild type neurons were phospho-JNK positive upon p75 activation compared with <20% of the nrif null neurons after p75 activation (Fig. 3B). Thus, the expression of NRIF is required for the stimulation of JNK by p75. We also investigated p75 regulation of this pathway by immunostaining for the transcription factor c-Jun. c-Jun is a well characterized downstream target of JNK, which up-regulates itself and accumulates in the nucleus following phosphorylation by JNK, thus it is often used to evaluate the activation of the kinase. Surprisingly, we found that the overall level of c-Jun was significantly reduced in the nrif–/– neurons and BDNF treatment still elicited some accumulation of the transcription factor in the nucleus (Fig. 3C). This result suggests that there is a mechanism by which the transcription factor is regulated that is independent of JNK. However, p75-mediated cell death does not require c-Jun (11.Palmada M. Kanwal S. Rutkoski N.J. Gustafson-Brown C. Johnson R.S. Wisdom R. Carter B.D. J. Cell Biol. 2002; 158: 453-461Crossref PubMed Scopus (108) Google Scholar), thus our results implicating NRIF in the activation of JNK are more relevant to the apoptotic pathway. Deletion of nrif Does Not Alter the Ability of p75 to Activate NF-κB—Another signaling system that regulates cellular viability and is under the influence of p75 is the NF-κB pathway. This transcription factor was shown to be activated in Schwann cells by NGF binding to p75 and to promote survival (25.Khursigara G. Bertin J. Yano H. Moffett H. DiStefano P.S. Chao M.V. J. Neurosci. 2001; 21: 5854-5863Crossref PubMed Google Scholar). Therefore, we evaluated the stimulation of NF-κB by neurotrophins in Schwann cells from nrif+/+ and –/– mice by a luciferase reporter assay (Fig. 4). There was no significant difference between the genotypes in the ability of p75 to activate this transcription factor. NRIF Expression Induces Apoptosis in Primary Cells—The observation that neurons do not undergo p75-mediated cell death or JNK activation in the absence of nrif suggested that this interactor mediates an apoptotic signal. Previous findings demonstrated that expression of NRIF in cell lines, such as 293 HEK cells, primarily caused cell cycle arrest (26.Benzel I. Barde Y.A. Casademunt E. Gene (Amst.). 2001; 281: 19-30Crossref PubMed Scopus (22) Google Scholar); however we considered the possibility that NRIF may activate cell death in primary, non-immortalized cells. To test this hypothesis, NRIF was expressed in primary Schwann cells, sympathetic neurons, or mouse embryo fibroblasts (MEFs). When mouse sympathetic neurons were infected with an adenovirus expressing GFP and NRIF bicistronically, ∼45% of the GFP-positive neurons were apoptotic, determined by DAPI staining of condensed or fragmented nuclei (Fig. 5A). In contrast, <10% of these neurons were apoptotic when infected with the GFP virus alone. Similarly, when primary rat Schwann cells or mouse embryo fibroblasts (MEFs) were infected with the NRIF virus, a significant increase in apoptosis was observed as compared with the GFP control. Similar results were seen in the absence of the adenovirus when the Schwann cells were transiently transfected with NRIF or GFP expression vectors (data not shown). These results demonstrate that expression of NRIF is sufficient to induce cell death in multiple primary cell types. The cell death induced by NRIF expression involved activation of caspase 3 and cytochrome c release from the mitochondria. As depicted in Fig. 5B, 24 h after transfecting rat Schwann cells with GFP-NRIF, the GFP-positive cells typically displayed a condensed or fragmented nucleus and were reactive to an antibody that recognizes the activated form of caspase 3. In many situations, it has been found that release of cytochrome c from the mitochondria precedes the activation of caspase 3. To determine if NRIF-mediated apoptosis involved cytochrome c release, Schwann cells were infected with adenovirus expressing NRIF or GFP, subjected to subcellular fractionation and the heavy membrane fraction, which is enriched with mitochondria, was evaluated for cytochrome c content by immunoblotting. At both 18 and 24 h (data not shown) after infection we observed a decrease in the amount of cytochrome c in the mitochondria from NRIF-expressing cells relative to those infected with adeno-GFP (Fig. 5C). Taken together, we conclude that NRIF is a pro-apoptotic molecule that induces the release of cytochrome c from the mitochondria and activates caspase 3. Given that p75 was not able to maximally activate JNK in the absence of NRIF, we considered the possibility that NRIF induced cell death by activating this kinase; however, over expression of NRIF in MEFs or Schwann cells (data not shown) did not significantly increase the levels of phospho-JNK (Fig. 5D). Thus, NRIF is necessary, but not sufficient for stimulating the kinase, as was previously suggested based on ectopic expression of NRIF in 293 HEK cells (24.Gentry J.J. Rutkoski N.J. Burke T.L. Carter B.D. J. Biol. Chem. 2004; 279: 16646-16656Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Both p75- and NRIF-mediated Apoptosis Are p53-Dependent—The tumor suppressor p53 has also been implicated in p75-mediated apoptosis, based on the ability of the inhibitor E1B to prevent the receptor induced death (13.Aloyz R.S. Bamji S.X. Pozniak C.D. Toma J.G. Atwal J. Kaplan D.R. Miller F.D. J. Cell Biol. 1998; 143: 1691-1703Crossref PubMed Scopus (260) Google Scholar). To directly determine whether the neuronal apoptosis induced by p75 activation requires p53, sympathetic neurons were isolated from p53+/+ and –/– mice and subjected to NGF withdrawal or p75 activation by BDNF, as above. Interestingly, although the p53–/– neurons were totally resistant to cell death induced by p75 activation (Fig. 6A), they underwent apoptosis as well as the wild type following NGF removal (Fig. 6B). Thus, these results demonstrate that, like NRIF, p53 is required for p75- mediated apoptosis but is dispensable for cell death occurring after NGF deprivation. To determine whether p53 was also required for NRIF-mediated cell death in these neurons, we cultured sympathetic neurons from p53 littermates and infected them with the GFP- or NRIF-expressing adenovirus and scored the number of apoptotic nuclei 48 h later. While the p53+/+ neuro
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