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J. Biol. Chem., Vol. 276, Issue 36, 33812-33820, September 7, 2001

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Characterization of a p75^NTR Apoptotic Signaling Pathway Using a Novel Cellular Model^*

Xin Wang, Johannes H. Bauer^§, Yong Li, Zhihong Shao, Firas S. Zetoune, Elena Cattaneo^¶, and Claudius Vincenz

From the  Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, the ^§ Department of Biochemistry, Free University, 14195 Berlin, Germany, and ^¶ Institute of Pharmacological Sciences, University of Milano, Via Balzaretti 9, 20133 Milan, Italy

Received for publication, November 21, 2000, and in revised form, July 10, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The p75 neurotrophin receptor (p75^NTR) belongs to the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In some cells derived from neuronal tissues it causes cell death through a poorly characterized pathway. We developed a neuronal system using conditionally immortalized striatal neurons, in which the expression of p75^NTR is inducibly controlled by the ecdysone receptor. In these cells p75^NTR induces apoptosis through its death domain in a nerve growth factor-independent manner. Caspases 9, 6, and 3 are activated by receptor expression indicating the activation of the common effector pathway of apoptosis. Cell death is blocked by a dominant negative form of caspase 9 and Bcl-X_L consistent with a pathway that involves mitochondria. Significantly, the viral flice inhibitory protein E8 protects from p75^NTR-induced cell death indicating that death effector domains are involved. A p75^NTR construct with a deleted death domain dominantly interferes with p75^NTR signaling, implying that receptor multimerization is required. However, in contrast to the other receptors of the family, p75^NTR-mediated apoptosis does not involve the adaptor proteins Fas-associated death domain protein or tumor necrosis factor-associated death domain protein, and the apical caspase 8 is not activated. We conclude that p75^NTR signals apoptosis by similar mechanisms as other death receptors but uses different adaptors and apical caspases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Apoptosis is widespread during development and disease states of the mammalian nervous system (1, 2). The high affinity neurotrophin receptors (Trk) as well as p75^NTR1 have been implicated in this process. Previous studies have shown that the low affinity NGF receptor p75^NTR as well as the high affinity NGF receptor TrkA are widely expressed throughout the central and peripheral nervous system of mammals (3). A few cell types only express p75^NTR but not TrkA, such as Schwann cells and oligodendrocytes (4, 5). In vitro culture of these cells and exposure to NGF leads to p75^NTR-induced apoptosis. Furthermore, the pro-apoptotic activity of p75^NTR has been documented in vivo. Retinal neurons and sympathetic neurons are killed by p75^NTR (6, 7). Transgenic expression of the intracellular domain of p75^NTR induced apoptosis in selected populations of central and peripheral neurons (8). On the other hand, p75^NTR has also been reported to elicit the opposite activity and increase survival leading to the generation, differentiation, and maintenance of distinct populations of neurons in mammalian nervous system through modification of TrkA signaling (9).

The neurotrophin family includes NGF, brain-derived neurotrophic factor, neurotrophin-3 (NT-3), NT-4/5, and NT-6. p75^NTR binds all neurotrophins with low affinity, but ligand-induced signaling has only been shown repeatedly for NGF (10) and in single reports for brain-derived neurotrophic factor (7) and NT4 (11).

NGF binding to p75^NTR has been reported to trigger NF-B, activate JNK, and release ceramide (12-14). p75^NTR-induced apoptosis is NGF-dependent in some systems but not others (6, 15). In immortalized cell lines a p75^NTR-mediated apoptotic response has been reported only in combination with nonspecific stresses like serum withdrawal, tamoxifen, or co-expression of molecules potentially involved in p75^NTR signaling (15-17).

As a death receptor (DR), p75^NTR belongs to the NGF/TNF receptor superfamily. To date, seven DRs have been identified that contain a conserved death domain (DD) (18). This domain has been shown to mediate apoptotic signaling by mediating homotypic interactions with adaptor proteins (19). Although the p75^NTR DD displays all the structural hallmarks of a DD, no homotypic interaction partners have been identified so far (20).

In contrast, the Fas and TNFR1 death pathways are well understood and involve receptor activation by their cognate ligands, FasL and TNF-, respectively. Upon activation, the adaptor proteins FADD and FADD/TRADD are recruited and activate caspase 8. Activated caspase 8 initiates proteolytic activities of other downstream caspases like caspase 3 and caspase 6, which then cleave specific substrates resulting in apoptotic morphology and DNA fragmentation (19).

The apoptotic signaling mechanism of p75^NTR is not understood in such detail. However, caspase inhibitors and Bcl-X_L inhibit the process suggesting that the downstream components are conserved (21, 22). The proteins SC-1, NRIF, NADE, ERKs, FAP-1, caveolin-1, NRAGE, as well as the GTPase RhoA have all been reported to bind the intracellular domain of p75^NTR and will potentially provide some insight into the signaling cascades triggered by this receptor (16, 23-29). However, it is currently not understood how any of these molecules are able to transmit the signal to the apoptotic effector machinery.

Cellular systems have been instrumental in elucidating signaling from other death receptors such as FAS and TNFR1. To undertake similar studies, we developed a cellular system as a tool to study p75^NTR-induced apoptotic signaling. p75^NTR receptor expression in ST14A cells was sufficient to induce cell death without the confounding effect of co-stimuli. ST14A cells are conditionally immortalized neurons derived from the striatum and retain the expression of multiple markers characteristic for neurons, but in addition they are highly transfectable and immortalized at the permissive temperature (30).

By using this system we show that the DD is essential for the initiation of apoptosis. Truncation mutants without DD are inactive and dominantly interfere with p75^NTR signaling. The similarities to other DRs also included the inhibition by the viral protein E8, activation of caspase 9, and multiple downstream caspases. However, caspase 8 was not activated, and FADD DN did not have any inhibitory effect. Hence, p75^NTR induces apoptosis in ST14A cells through a similar molecular mechanism to that of other DRs but via different adaptors and apical caspases.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents and Antibodies-- Zeocin and ponasterone were obtained from Invitrogen (Carlsbad, CA); hygromycin B was from Roche Molecular Biochemicals; LipofectAMINE was from Life Technologies, Inc.; and NGF was from Harlan Bioproducts (Madison, WI). Caspase substrates were from Stratagene (La Jolla, CA). The BrdUrd kit was from BD PharMingen (San Diego, CA). Propidium iodide (PI), poly-L-lysine hydrobromide, protein G-Sepharose, -FLAG M2 antibody, and anti-mouse IgG fluorescein isothiocyanate conjugate (Fc-specific) were from Sigma. -NGF receptor polyclonal antibody was from Promega (Madison, WI). Secondary antibodies and ECL reagents were from Amersham Pharmacia Biotech.

Cell Lines, Expression Vectors, and Transfections-- ST14A are striatal neurons conditionally immortalized by transfection with a temperature-sensitive form of the SV40 large T-antigen (30). ST14A and 293 human embryonic kidney cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and penicillin/streptomycin in 5% CO₂ at 33 °C (for ST14A cells) or 37 °C (for 293 cells). Human neuroblastoma cells, SK-N-BE, were grown at 37 °C in minimum Eagle's medium/Ham's F-12 nutrient mixture (1:1) with the same additions as for ST14A cells. Schwann cells were isolated from sciatic nerve of neonatal rats, and single cell suspension was achieved by trypsin and collagenase and cultured with 10% fetal bovine serum, 6 mM L-glutamine, penicillin/streptomycin, 20 µg/ml pituitary extract, and 2 µM forskolin on poly-L-lysine-coated tissue culture dishes at 37 °C. A431 carcinoma cells were grown in Dulbecco's modified Eagle's medium/F-12 (1:1) containing 2.0 mM glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 0.25 mg/ml amphotericin B, and 5% fetal bovine serum at 37 °C.

The p75^NTR constructs shown in Fig. 1 were obtained by PCR and cloned into the ecdysone-inducible mammalian expression vector pIND-Hygro (Invitrogen) using a human p75^NTR plasmid kindly provided by Moses V. Chao (New York University). All constructs were verified by sequencing and ponasterone-inducible expression in 293 cells. For stable expression, p75^NTR constructs were transfected together with the ecdysone receptor plasmid pVgRXR. Cells expressing p75NTR (ST14A-p75^ind) were selected by growth in hygromycin B (0.625 mg/ml) and zeocin (0.625 mg/ml) and screened for expression by immunoblotting.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Diagram of human p75NTR constructs used in this study. The amino acids at the ends of the domains and of the constructs are numbered.

RT-PCR-- Total RNA (1 µg) was isolated from the different cell lines using Trizol reagent (Life Technologies, Inc.). After reverse transcription, cDNA was amplified using superscript one-step RT-PCR system (Life Technologies, Inc.). For RT-PCR, the following primers were used: rat p75^NTR, 5'-AGCCAACCAGACCGTGTGTG-3' and 5'-TTGCAGCTGTTCCACCTCTT-3' (31); rat TrkA, 5'-GCTGACCAATGAGACCATGCGGCAT-3' and 5'-GTGAGCAGCTCTGCCTCACGATGG-3' (31); rat GAPDH, 5'-ATGGTGAAGGTCGGTGTCAACGGA-3' and 5'-TTACT- CCTTGGAGGCCATGTAGGC-3'.

MTS Assay-- The assay was performed according to the manufacturer's instructions (Promega, Madison, WI). About 5 × 10⁴ cells grown in a 96-well plate in 100 µl of medium were either left untreated or treated with ponasterone. Twenty µl of combined MTS/PBS solution was added, and the cells were put in the incubator for 1 h before reading the absorbance at 490 nm.

DNA Fragmentation Assay-- About 5 × 10⁶ ST14A cells treated with ponasterone for different times were harvested by DNAZol (MRC, Ohio). DNA was purified by EtOH precipitation. The precipitate was dissolved in H₂O, digested for 30 min at 37 °C with 20 µg/ml DNase-free RNase, and analyzed by electrophoresis on a 1% agarose gel.

Measuring p75^NTR Expression by FACS Analysis-- About 5 × 10⁶ ST14A cells were treated with ponasterone or left untreated. Attached cells were harvested in 135 mM potassium chloride, 15 mM sodium citrate at 37 °C for 5 min. The resulting cell suspensions were pooled with cells in the supernatant, washed with 1% heat-inactivated fetal bovine serum in PBS, blocked with 3% bovine serum albumin in PBS for 30 min, and incubated with -FLAG or -p75^NTR antibody (1:100) for 1.5 h and fluorescein isothiocyanate-conjugated secondary antibody (1:2000) for 1 h. All processing was performed at 4 °C. The cells were analyzed on a Beckman flow cytometer.

Immunoprecipitation and Immunoblotting-- About 5 × 10⁶ cells were collected in lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA with 5 mM Na₂VO₄, protease inhibitor mixture (Roche Molecular Biochemicals), 0.2 mM phenylmethylsulfonyl fluoride) on ice and directly analyzed by Western blot or immunoprecipitated after clearing at 10,000 × g for 10 min. The soluble lysates were incubated with the relevant antibodies for 3 h, and immune complexes were harvested with protein G-Sepharose beads for 1 h, followed by three washes in lysis buffer, and boiling in sample buffer. Eluted proteins were separated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blot (1:1000 dilution of primary and 1:10000 of secondary antibodies).

Microscopic Determination of Cell Death-- About 1 × 10⁶ ST14A-p75^ind cells were grown on glass coverslips coated with gelatin and co-transfected with plasmids expressing the indicated proteins (1 µg) and GFP as a transfection marker (0.25 µg). After transfection, cells were treated with ponasterone for 48 h, washed with PBS, fixed with 4% paraformaldehyde, incubated for 10 min in 0.5 µg/ml PI, and washed with PBS. Transfected (green) cells were scored by fluorescence microscopy for chromatin condensation, and nuclear fragmentation was revealed by intense PI staining.

TUNEL Assay-- About 1 × 10⁶ ST14A-p75^ind cells were transfected as above and treated with ponasterone for 48 h. Attached cells and matching supernatant were pooled, fixed with 4% paraformaldehyde, and submitted to terminal deoxytransferase-mediated BrdUTP nick-end labeling (TUNEL) using phycoerythrin-conjugated anti-BrdUrd monoclonal antibody flow cytometer following the instructions of the manufacturer. Labeled cells were analyzed on a Beckman flow cytometer.

Caspase Activity Assays-- Cell extracts and enzyme assays were performed as described previously (32). Briefly, 5 × 10⁶ cells were harvested in PBS and homogenized with a Dounce homogenizer in extraction buffer (10 mM KCl, 1 mM dithiothreitol, 5 mM EGTA, 25% glycerol, 1 µg/ml each of leupeptin, aprotinin, soybean trypsin inhibitor, and pepstatin, 0.2 mM phenylmethylsulfonyl fluoride in 10 mM Hepes, pH 7.4) with 40 strokes. Twenty µg of lysate proteins in a 100-µl final volume were incubated with 0.2 mM of the following caspase substrates at 37 °C for 3 h: caspase 1, Ac-YVAD-AFC; caspase 3, Ac-DEVD-AFC; caspase 6, Z-VEID-AFC; caspase 8, Ac-IETD-AFC; and caspase 9, Ac-LEHD-MCA. Caspase substrates incubated in non-ponasterone-treated extracts were used as control. Released AFC or MCA was read in a cytofluor II fluorescence multiwell plate reader (excitation at 400 nm and emission at 505 nm for AFC-labeled substrates or excitation at 360 nm and emission at 460 nm for the MCA substrate).

NGF Measurements-- The enzyme-linked immunosorbent assay kit from Promega (Madison, WI) was used to determine NGF in the media according to the instructions of the manufacturer. Cells were incubated in serum-free media supplemented with 1% bovine serum albumin for 24 and 48 h prior to the assay.

Statistical Analysis-- StatView software was used to calculate significant differences by analysis of variance algorithm. Error bars indicate S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

p75 Induces Cell Death in ST14A Cells-- Multiple functional studies on p75^NTR have been performed using primary neuronal cells and transgenic or knockout models (33). These studies show that p75^NTR engages multiple and sometimes opposing signaling pathways, depending on the cell type, microenvironment, and stage of development (10). Invariably, cellular systems developed to study p75^NTR function and mechanisms show only a subset of the responses attributed to p75^NTR signaling.

In search of a model for the apoptotic signaling of p75^NTR, we investigated the ST14A cell line. These cells are derived from the striatum of embryonic day 14 rats and are conditionally immortalized by expression of the temperature-sensitive mutant of the SV40 large T antigen (30). ST14A cells acquire antigenic and electrophysiological properties characteristic of mature neurons and become postmitotic at the non-permissive temperature (39 °C) (34). Because these neuronal cells are susceptible to a variety of apoptotic stimuli, including the death receptor TNFR1 (35), we investigated their responses to p75^NTR.

Stable ST14A expressing p75^NTR lines (ST14A-p75^ind) were established using an ecdysone-inducible system to avert problems with the pro-apoptotic activity of p75^NTR during the selection process. This two-plasmid system puts the gene of interest under the control of the Ecdysone/glucocorticoid response element promoter. The ecdysone receptor is transcribed from the co-transfected pVgRXR plasmid, and expression of the gene of interest is induced by addition of the ecdysone homologue ponasterone. Expression of the two NGF receptors in parental ST14A as well as lines carrying an expression vector for p75^NTR was assayed by RT-PCR (Fig. 2A). Parental ST14A cells were negative for p75^NTR but expressed similar levels of TrkA as the carcinoma cell line A431. Exposing these cells to ponasterone had no effect on the levels of NGF receptors (lanes 3 and 4). Analysis of the ST14A-p75^ind cells revealed expression of p75^NTR RNA both in a pool and two randomly selected clones (lanes 6-11). Robust induction of p75^NTR by ponasterone was observed after 15 PCR cycles (lanes 5 and 6). Amplification of the RT product for 30 cycles also yielded a band in the absence of ponasterone indicating some leakiness of the promoter (lanes 7 and 9). The levels of expression were lower than endogenous p75^NTR levels in Schwann cells (lane 11). TrkA levels remained unchanged in the transfectants with or without ponasterone. These results indicate that the ST14A clone used here did not express endogenous p75^NTR and therefore represent a convenient null background for the introduction of wild type or mutants.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Expression of p75NTR in ST14A cells is cytotoxic. A, p75NTR and TrkA mRNA expression were analyzed in ST14A cells. RT-PCR was performed on total RNA extracted from ST14A cells (lanes 3 and 4) and cells stably transfected with p75NTR (lanes 5-10). Lanes 5 and 6 and 9 and 10 are single clones, and lanes 7 and 8 are pooled p75NTR transfectants. Where indicated, ponasterone treat- ment was for 36 h. RT-PCRs were amplified for 15 or 30 cycles as indicated using the gene-specific primers shown on the right. Negative controls (N. Ctrl.): SK-N-BE for p75NTR; RNase-treated ST14A sample for TrkA. Positive controls (P. Ctrl.): Schwann cells for p75NTR; A431 carcinoma for TrkA and GAPDH; GAPDH mRNA was selected as internal control. B, ST14A-p75ind cells were treated with ponasterone for 48 h at 33 °C. The transfectants were assayed at the indicated passage numbers. Viability of the cells was determined by the MTS assay and compared with controls that were treated identically except for the addition of ponasterone. Parental ST14A cells were included as the control for nonspecific ponasterone effects. The values are mean ± S.E. of three determinations. Significant killing compared with non-treated control is indicated by * (p < 0.05). C, cell surface expression of FLAG-p75NTR in passage 2 and 40 cells was measured by FACS after overnight incubation with ponasterone.

Addition of ponasterone to ST14A-p75^ind cells produced dramatic cell killing as demonstrated by measuring viability with the MTS assay (Fig. 2B). After a 2-day treatment with ponasterone at 33 °C about 75% of mitochondrial activity was lost in freshly established ST14A-p75^ind compared with the untreated cells. The cells detach from the substrate, round up, and develop cell membrane blebs during the treatment (not shown). However, ST14A-p75^ind cells that were kept in culture for extended periods gradually became resistant to ponasterone at 33 °C (passage 40). Ponasterone was not cytotoxic when added to parental ST14A cells.

To demonstrate expression of the exogenous protein on the cell surface, FACS analysis was performed on p75^NTR-FLAG transfectants (Fig. 2C). The epitope tag in this construct is engineered immediately C-terminal to the signal peptide (Fig. 1). Non-permeabilized cells were stained with -FLAG antibodies with or without ponasterone treatment. Fluorescence in the absence of ponasterone was identical to the background control. After overnight incubation with ponasterone, increased staining of cells with -FLAG was measured. In contrast, late passage cells did not show any expression at 33 °C. However, at 39 °C expression as well as sensitization to cell killing by ponasterone was restored (not shown). To avoid these confounding factors of prolonged culture all subsequent experiments were performed on cells passaged less than 20 times unless indicated.

Because in some systems NGF is able to induce cell death by binding to p75^NTR, we investigated whether addition of this neurotrophin affects the cell death response in ST14A-p75^ind. The loss of viability of ST14A-p75^ind upon ponasterone treatment was not affected by incubation in serum-free media indicating that the process was not controlled by serum-derived factors (Fig. 3A). Similarly, cell killing induced by ponasterone was neither enhanced nor inhibited by the addition of NGF (10 ng/ml) as measured by MTS assay (Fig. 3A). To investigate a possible autocrine mechanism, NGF levels in the media of parental and ST14A-p75^ind were measured by enzyme-linked immunosorbent assay. After 48 h of incubation of parental ST14A cells 75.25 ± 19 and 39.38 ± 17.41 pg/ml NGF was detected in the absence and presence of ponasterone, respectively. The p75^NTR transfectants produced similarly low levels of NGF (29.5 ± 4.91 pg/ml). These levels of NGF are in the picomolar range well below the nanomolar concentrations required for p75^NTR activation. Therefore, a NGF autocrine mechanism cannot account for the p75^NTR-induced cell death in ST14A-p75^ind.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   p75NTR cytotoxicity is not modulated by NGF and requires the death domain. A, ST14A-p75ind cells were treated with ponasterone for 48 h at 33 °C and the indicated culture conditions. Viability was measured with the MTS assay. Parental ST14A cells and transfectants expressing p75NTR deletions were assayed similarly. Mean values ± S.E. of three experiments are shown. Significant killing by ponasterone (Pon.) is indicated by * (p < 0.05). B, expression of the FLAG-tagged constructs was assayed by FLAG immunoprecipitation (I.P.) and Western blot (W.B.). For comparison with endogenously expressed p75NTR, lysates containing equal amounts of total protein were immunoprecipitated with -p75NTR and analyzed on a Western blot with the same antibody. S.C., rat Schwann cells, not treated. Arrows indicate p75NTR bands.

NF-B activation in ST14A-p75^ind cells was also measured. No significant induction of this pathway could be observed using luciferase reporter assays (not shown), suggesting that p75^NTR cannot activate NF-B in these cells or that the strong pro-apoptotic response masks other signals that potentially emanate from this receptor.

The intracellular domain of p75^NTR contains a well conserved DD whose role in initiating apoptosis has been disputed (21, 36). To address this issue in the ST14A system, we established ST14A-p75^ind lines expressing FLAG-tagged full-length and deletion mutants (Fig. 1). Deletions of the DD (DD) and the whole intracellular domain (ICD) were tested. Expression of both the DD and ICD had no effect on cell viability similar to parental ST14A cells (Fig. 3A). Levels of expression were measured by immunoblotting with -FLAG antibodies (Fig. 3B). Ponasterone induced the expression of a doublet band at ~75 kDa in the FL transfectants. This is the molecular weight reported for the glycosylated form of endogenous p75^NTR (37). Immunoprecipitation with p75^NTR-specific antibodies revealed that the levels of p75^NTR expressed were slightly lower than endogenous p75^NTR in Schwann cells. A similar level of expression was seen when lysates from cells expressing the DD and ICD constructs were probed together with the full-length p75^NTR lysates. Doublet bands at ~65 and 45 kDa were induced by ponasterone with the DD and ICD, respectively. The transfectants expressing the truncation mutants were also analyzed by FACS for cell surface expression using the -p75^NTR antibody. More than 75% of the cells stained positive after overnight incubation with ponasterone (not shown). Therefore, the failure of the DD and ICD to induce apoptosis is due to the inability of these mutants to signal cell death and not due to lack of expression or inappropriate cellular localization. In these cells, therefore, deletion of the DD impairs the ability of p75^NTR to signal cell death. Shown here are the results from transfection pools, but the analysis of 7 DD and 3 ICD expressing clones produced identical results.

p75^NTR Induces Apoptosis-- The size distribution of DNA recovered from ST14A cells was analyzed in order to establish whether p75^NTR-induced cell death proceeded by an apoptotic mechanism (Fig. 4A). Treatment of the ST14A-p75^ind cells with ponasterone induced degradation of DNA into a pattern of ~200-base pair periodicity that was particularly distinct after 48 h when most cells have acquired apoptotic morphology. This degradation was not observed without ponasterone treatment. Such an oligonucleosomal pattern is a prominent feature of apoptosis and therefore indicates that p75^NTR is able to activate an apoptotic pathway. Consistent with the observation that the DD and ICD were not cytotoxic, no DNA degradation was observed upon ponasterone induction of those cell lines.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Expression of p75NTR induces apoptosis. A, the p75NTR transfectants were treated with ponasterone (Pon.) for the hours indicated. Total DNA was extracted from an equal number of cells and separated on an agarose gel. B, approximately 5 × 106 ST14A-p75ind transfectants were left untreated or treated with 5 or 15 µM ponasterone for 48 h. All cells were harvested and homogenized. The DD and ICD cell lines were treated similarly and assayed for caspase-9 activity. Equal amounts of protein were incubated with 0.2 µM of caspase substrates at 37 °C for a time course of 3 h. The fluorescence intensities were calibrated with free AFC and MCA. Substrate conversion rates were calculated using the slope of the time course and corrected by subtracting values obtained from the untreated extracts. The values are mean ± S.E. of three independent experiments. The inset shows the caspase 8 control experiment. Cells were transiently transfected with 1 µg of pcDNA3.1 or 1 µg of TNFR1 for 24 h, or treated with 100 ng/ml TNF plus 10 µM cycloheximide for 6 h. The lysates were assayed with the caspase 8 substrate.

The appearance of an oligonucleosomal ladder indicated that DNases were activated by p75^NTR. Because the activity of apoptotic DNases is regulated by caspases (38), we attempted to identify which caspases participate in the execution of p75^NTR-induced apoptosis. Cellular extracts were prepared after treatment with ponasterone, and caspase activities were measured using specific fluorogenic tetrapeptide substrates. Robust induction of caspase 9 activity as well as of the downstream caspases 3 and 6 was observed. The caspase 1 substrate was also cleaved efficiently in the ponasterone-activated extract (Fig. 4B). Significantly, no caspase 8 activity was induced. To verify that caspase 8 can be activated and its activity measured by the assay, a control experiment was performed (inset). The cells were either transfected with TNFR1 or treated with TNF- in the presence of cycloheximide. The caspase 8 substrate was cleaved when added to these extracts demonstrating that the assay detects activation of this caspase in this cell line. Therefore, the absence of caspase 8 activity in ST14A-p75^ind cells suggests that caspase 8 is not involved in p75^NTR-induced apoptosis and implies that p75^NTR signal transduction activates a different apical caspase than FAS or TNFR1 in ST14A cells. The control ST14A cell line expressing ICD p75^NTR did not induce caspase 9 activity upon ponasterone treatment, consistent with the inability of this truncation mutant to induce apoptosis. Moreover, cells expressing the DD construct did not respond to ponasterone treatment with increased caspase activity either, further documenting the proapoptotic activity of this domain.

p75^NTR-induced Apoptosis Is Repressed by General and DR-specific Inhibitors-- The ST14A system made it feasible to delineate which components of the apoptotic machinery are involved in p75^NTR-mediated apoptosis. Several anti-apoptotic molecules with specific molecular targets that occur naturally have been identified or have been engineered. The effect of these inhibitors on the p75^NTR apoptotic pathway was studied (Fig. 5). ST14A-p75^ind cells were co-transfected with a plasmid expressing the indicated inhibitor and GFP as a transfection marker. The PI staining pattern of the transfected cells was evaluated by fluorescence microscopy after 24 h of ponasterone treatment (Fig. 5A). Vector and GFP alone induced a background of an apoptotic staining pattern of ~7%. Treatment with ponasterone increased this value to 22%, which is an underestimate as floating cells are lost to analysis in this assay.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   General and DR-specific inhibitors repress p75NTR-induced apoptosis. A, p75NTR-FL stable lines were co-transfected with GFP expression vector and the plasmid expressing the indicated inhibitor. The cells were treated with ponasterone (Pon.) for 48 h and nuclei stained with PI. The PI staining pattern of transfected cells was evaluated by fluorescence microscopy. The number of transfected cells with apoptotic PI staining over the number of total counted GFP-positive cells is given. At least two independent experiments were evaluated. Significant inhibition compared with ponasterone-treated control is indicated by * (p < 0.05). Inset, parental ST14A cells were transfected with TNFR1, GFP, TRADD DN, and FADD DN constructs. Apoptotic DNA staining pattern was evaluated after 48 h as in A. B, p75NTR-FL-FLAG stable lines were transfected similarly and processed for TUNEL assay. Transfected (GFP) and TUNEL-positive cells were quantitated by FACS. As an example, the fluorescence intensity data from untreated (Ctrl.), vector control (Pon.), and E8-transfected cells is shown. The quantitation of the percentage of double positives is given. Results shown are from three separate experiments. Significant inhibition compared with ponasterone-treated control is indicated by * (p < 0.05).

We tested E8 and FADD DN as inhibitors specific for DRs (39-42). Expression of E8, an equine herpesvirus protein consisting of two death effector domains, effectively inhibited the apoptotic response. In contrast, transfection of FADD DN, a construct containing only a DD and missing its death effector domain, did not reduce the apoptotic response. This is surprising, because FADD DN, like E8, inhibits signaling from other DRs. TRADD DN, an inhibitor specific for TNFR1-induced apoptosis (43), also had no effect.

To ascertain that the dominant negative forms of FADD and TRADD were active in ST14A cells, a control experiment was performed using transfection of TNFR1 as the pro-apoptotic stimulus (Fig. 5A, inset). Expression of both molecules inhibited TNFR1-induced cell death as effectively as observed in non-neuronal cells.

To establish whether mitochondria were involved in mediating or amplifying the p75^NTR signal, we tested caspase 9 DN and Bcl-X_L. Anti-apoptotic Bcl-2 homologues act by inhibiting the release of pro-apoptotic molecules, such as cytochrome c, from mitochondria, whereas caspase 9 DN inhibits the ensuing caspase 9 activation at the apoptosome (44, 45). Caspase 9 DN and Bcl-X_L were both effective inhibitors and reduced the number of cells with a dense PI staining pattern to almost background levels.

CrmA is a viral caspase inhibitor with highest affinity for caspase 1, followed by caspase 8 (46). CrmA was also able to mediate substantial inhibition of cell death, which is consistent with our finding that a caspase 1-like activity is induced (Fig. 4).

To obtain independent confirmation of these results, we analyzed ST14A-p75^ind cells using TUNEL as an alternative method to measure apoptosis. Cells were transfected, and p75^NTR was induced as above, but analysis was performed by labeling DNA ends with BrdUrd. The amount of apoptosis was determined by measuring the TUNEL and GFP double-positive cells (Fig. 5B). Almost identical results were obtained using this unbiased approach as with the microscopic evaluation.

p75^NTR Signaling Requires Receptor Multimerization-- A prerequisite for DR signaling is the oligomerization of the receptor. The prototype DR, TNFR1, exists as a trimer. Similarly Fas is also oligomerized (47, 48). Recently it has been shown that TNFR1 and Fas already pre-exist on the cell surface as oligomers before ligand binding (49, 50). Because of this multimerization requirement, expression of deleterious receptor mutations leads to a DN phenotype as long as they still multimerize (50). To investigate further whether p75^NTR has similar requirements for receptor homo-oligomerization as other DRs, we tested the C-terminal truncation mutants for a DN effect (Fig. 6A). The DD and the ICD p75^NTR constructs (Fig. 1) were co-transfected into ST14A-p75^ind cells together with GFP marker. Wild type p75^NTR was induced by the addition of ponasterone. Microscopic evaluation showed that p75^NTR without a DD acted as a strong DN and inhibited apoptosis as effectively as E8. Deletion of the entire intracellular domain did not produce a molecule with protective properties. Western blot analysis revealed that induction of p75^NTR was similar in all transfections indicating that the inhibitors affected directly p75^NTR signaling and not its expression. Co-immunoprecipitation experiments were performed to assess the binding potential of the deletion constructs to full-length p75^NTR (Fig. 6B). FLAG-tagged p75^NTR full length and deletion constructs were co-expressed in 293 cells together with full-length p75^NTR-GFP. FLAG immunoprecipitation and Western blotting with -GFP revealed that full-length, DD, and ICD p75^NTR were all able to associate with p75^NTR-FL-GFP. p75^NTR-GFP did not associate nonspecifically with FLAG beads. Therefore, p75^NTR, like other receptors of this family, was able to self-associate through the extracellular domain, and incorporation of signaling defective mutants into the complex leads to its inactivation.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Dominant interference of a p75NTR mutant defective in apoptotic signaling. A, p75NTR-FL stable lines were co-transfected with GFP vector and the plasmids of mutants constructs or E8. Cells were treated with ponasterone for 72 h. Quantitation of apoptotic DNA condensation of the transfected cells was evaluated after PI staining. The results are from three experiments. Significant inhibition is indicated by * (p < 0.05). Expression of p75NTR was monitored by FLAG immunoprecipitation and Western blotting. B, 293 cells were co-transfected with the indicated p75NTR-FL-GFP construct and FLAG-tagged FL or mutant constructs. Lysates (Lys.) were immunoprecipitated (IP) and analyzed by GFP Western blot (W.B.). The gel was reprobed with FLAG to confirm expression of the FLAG-tagged constructs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A New Cellular Model-- The ST14A model presented here is the only model where a strong apoptotic response is elicited by p75^NTR alone without any additional stimuli. ST14A cells are effectively killed by induced expression of p75^NTR. Interestingly, addition of NGF had no effect. Endogenous levels of NGF secreted into the media are well below the affinity of p75^NTR excluding an autocrine mechanism. These observations are in support of the hypothesis that p75^NTR signals in the absence of ligand in some cells (15). ST14A-p75^ind cells were not rescued by the addition of NGF, suggesting that addition of ligand does not negate the pro-apoptotic activity of p75^NTR. TrkA on ST14A cells is also insufficient to prevent apoptosis, even though TrkA was expressed on ST14A-p75^ind cells and was activated by NGF as assayed by measuring NGF-induced tyrosine autophosphorylation (not shown).

The large T-antigen has been reported to inhibit apoptosis by death receptors, serum starvation, and caspase-1 activation, raising the possibility that the immortalization of ST14A will interfere with apoptotic signaling (51-53). The observation, however, that p75^NTR-induced cell death is unimpeded at the non-permissive temperature of 39 °C implies that the pro-apoptotic responses are independent of the large T-antigen. The development of resistance after extensive passaging of the cells is potentially mediated by the large T-antigen as sensitivity and expression were regained upon shifting to the non-permissive temperature. Further studies are needed to evaluate the role of the large T-antigen for the development of resistance.

Expression of the receptor in ST14A-p75^ind cells is comparable to endogenous levels of p75^NTR in Schwann cells (Fig. 3B) suggesting that our system is relevant to in vivo situations. The inducible system permits regulatable expression of p75^NTR and induction of cell death. It is therefore less susceptible to artifacts during the selection of stable lines, which is also confirmed by the uniformity of the ponasterone effect in single clones versus pools. ST14A-p75^ind cells therefore represent an example of a neuronal cell type where both NGF receptors are expressed and p75^NTR signals cell death in a dominant and ligand-independent manner. Similar p75^NTR dominant signaling has also been observed in vivo in peripheral nerves where the ICD of p75^NTR is capable of inducing cell death, even in the presence of neurotrophic support and TrkA (8).

The Apoptotic Signaling Mechanism of p75^NTR-- Deletion mutagenesis shows that the DD of p75^NTR is required for the induction of apoptosis (Fig. 3). The DD construct not only is unable to induce apoptosis but also interferes dominantly with apoptosis induced by wt-p75^NTR (Fig. 6). This indicates that cell death induction by p75^NTR is similar to other DRs, which all use this domain to recruit DD containing adaptor molecules (54). Extensive searches for p75^NTR interacting molecules have identified nine proteins that interact with the intracellular domain of p75^NTR (12, 16, 23-29). None of these proteins, however, contain a DD, and NADE is the only protein implicated in cell death that interacts with the DD of p75^NTR (25). The p75^NTR-DD structure contains all the marks of a typical DD and falls well within the structural variation observed within this family. No features have been identified that would prevent this domain to function as a homotypic interaction domain (55). The fact that the isolated p75^NTR DD does not form homodimers in solution has been used for the argument that this DD does not provide an interaction surface. However, other death domains with known binding partners also do not form homodimers or heterodimers in solution when expressed as isolated domains (56). Therefore, homotypic interactions partners for the p75^NTR DD may still be identified.

Experiments performed in sensory neurons have indicated that cell death can be induced by truncated p75^NTR without a DD (21). In this system a membrane-anchored juxtamembrane domain was sufficient to induce cell death (36). Considering the multitude of effects p75^NTR elicits, it is possible that sensory neurons signal cell death by a different mechanism than ST14A cells.

The equine herpesvirus E8 is a strong inhibitor of p75^NTR-induced apoptosis (Fig. 5). E8 acts by precluding the recruitment of caspase 8 to the death-inducing signaling complex (DISC) (39-42). Since we were unable to detect caspase 8 activation, we hypothesize that a different caspase is recruited by p75^NTR in ST14A cells (Fig. 4B). In support of this hypothesis, Gu et al. (22) reported that caspase 8 was not processed by NGF-triggered apoptosis in oligodendrocytes. This is further confirmed by the inability of FADD DN to interfere with p75^NTR signaling. Similar to E8, FADD DN inhibits recruitment of both caspase 8 and caspase 10 to the DISC (57). The involvement of different apical caspases and adaptor proteins is furthermore indicated by the observation that a fusion protein between the extracellular domain of Fas and the intracellular domain of p75^NTR is not able to kill cells that readily die by expression of Fas (58). Nonetheless, the E8 results indicate that death effector domain interactions are essential for p75^NTR signaling, much as they are needed for signaling from the other DRs. However, the proteins containing these death effector domains still need to be identified.

Cleavage of the caspase 1 substrate was induced by p75^NTR expression. The main role of caspase 1 is to cleave pro-interleukin-1 and not to mediate apoptosis (59). However, caspase 1 has the same substrate specificity as caspases 4 and 5, and they are both capable of inducing apoptosis (60, 61). The effective inhibition observed with CrmA confirms the involvement of this group of caspases.

Cells have been subdivided into type I or type II groups depending on the apoptotic mechanisms induced by Fas (62). Type I cells have high levels of DISC components, and apoptosis is not inhibited by Bcl-X_L. Type II cells generate a weaker DISC activity that requires the mitochondrial amplification loop to induce apoptosis, and Bcl-X_L protects in these cells. Given the inhibition by Bcl-X_L and caspase-9 DN the p75^NTR-ST14A cells described here belong to the type II group of cells.

Similar to other DRs, DD-p75^NTR is defective in signaling and acts as a DN, implying that multimerization of p75^NTR is required for signaling. The ICD, however, is able to multimerize but does not inhibit signaling. Possibly, the extracellular domain is unable to affect the conformation of the intracellular domains of its full-length interaction partners or a mutated intracellular fragment is required to interfere with the assembly of a functional receptor signaling complex. p75^NTR has also been shown to interact functionally and physically with the high affinity neurotrophin receptors (reviewed in Ref. 10). These heterotypic interactions are mediated by both the intracellular and the extracellular domains of both receptors. Interestingly, deletion of the ICD leads to an enhancement of TrkA basal signaling (63). This suggests that pro-apoptotic signaling through homotypic interactions of p75^NTR involves different p75^NTR domains than anti-apoptotic signaling through heterotypic interactions with the Trk receptors.

The primary sequence is not conserved well enough in the DR family to be able to identify a pre-ligand binding assembly domain on p75^NTR that has been shown on Fas and TNFR1 (49, 50). However, the ability of the ICD mutant to interact with p75^NTR-FL indicates that the extracellular domain is important for homotypic interactions.

Our results show that p75^NTR apoptotic signaling has similarities with other DRs. The ST14A model does not recapitulate all p75^NTR signals, but its strong apoptotic response provides the means to study DD-dependent signaling from this receptor. In particular, detailed structure guided mutational studies will provide further information on the DD interaction interface necessary to generate the apoptotic signal. This information will be valuable for evaluating which of the multiple p75^NTR-interacting proteins participate in the DD-dependent signaling and in the rational design of specific inhibitors. The cellular model presented here is suited to evaluate the pharmacological potential of small molecules or engineered proteins. The specificity of these reagents can also be assessed as the cells are susceptible to killing by other DRs such as TNFR1.

Even though we developed ST14A-p75^ind primarily as a tool for mechanistic studies, results obtained with cells are likely to be directly applicable to striatal neurons in vivo. The ST14A clone used in this study did not express endogenous p75^NTR (Fig. 2) contrary to a previous report (30), indicating clonal variability. Expression of p75^NTR in the rat striatum in vivo has only been shown in cholinergic neurons (64). However, the majority of the striatal neurons are positive for -aminobutyric acid expressing DARPP-32. ST14A cells also express DARPP-32 and are therefore more similar to the bulk of the striatal neurons (34). Expression of p75^NTR in DARPP-32-positive cells is not observed during development. However, ischemia has been shown to induce p75^NTR expression in the resistant cholinergic striatal neurons (65). In addition, striatal neurons have been shown to undergo apoptosis after transient focal ischemia (66). Possibly, expression of p75^NTR also occurs in the neurons positive for -aminobutyric acid early in the course of ischemia but are rapidly killed similarly to the ST14A cells presented here. The development of specific inhibitors for p75^NTR is needed to ascertain the therapeutic benefit of modifying the activity of p75^NTR for a variety of disease states. Multiple studies have indicated a possible role of this receptor in acute neuronal injuries and progressive neurodegenerative disorders. p75^NTR expression is up-regulated after axotomy or neuronal injury, and p75^NTR antisense oligonucleotides have been shown to reduce the damage (67-69). The -amyloid peptide that is accumulating as extracellular deposits during the progression of Alzheimer's disease has been shown to bind and activate p75^NTR (70, 71). The cellular model presented here will be a valuable tool for mass screening of anti-apoptotic compounds that may have a benefit to treat these and perhaps other neurological disorders where p75^NTR has been implicated.

	ACKNOWLEDGEMENTS

We thank Eva L. Feldman and James Russell for providing Schwann cells, Yoram Milner for providing A431 carcinoma cells, and Yuseef Namy for help in preparing the figures.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant ES08111 and Department of Defense Grant DAMD 17-96-6085.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, Rm. 7510 MSRB I, Box 0602, 1150 West Medical Center Dr., Ann Arbor, MI 48109. Tel.: 734-647-9551; Fax: 734-764-4308; E-mail: vincenz@umich.edu.

Published, JBC Papers in Press, July 12, 2001, DOI 10.1074/jbc.M010548200

	ABBREVIATIONS

The abbreviations used are: p75^NTR, p75 low affinity neurotrophin receptor; FL, full length; DD, death domain; DR, death receptor; DN, dominant negative; ICD, intracellular domain; NGF, nerve growth factor; PI, propidium iodide; FADD, Fas-associated death domain protein; TRADD, tumor necrosis factor-associated death domain protein; TNFR, tumor necrosis factor receptor; DISC, death-inducing signaling complex; RT-PCR, reverse transcriptase-polymerase chain reaction; NT, neurotrophin; BrdUrd, bromodeoxyuridine; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; TUNEL, terminal deoxytransferasemediated BrdUTP nick-end labeling; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AFC, 7-amino-4-trifluoromethyl coumarin; MCA, 4-methyl-coumayl-7-amide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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