Apoptosis induced by p75NTR overexpression requires Jun kinase-dependent phosphorylation of Bad

Abstract

The p75 neurotrophin receptor (p75NTR), a member of the tumor necrosis factor receptor superfamily, facilitates apoptosis during development and after injury to the CNS. The signaling cascades activated by p75NTR that result in apoptosis remain poorly understood. In this study, we show that overexpression of p75NTR in primary cortical neurons, in pheochromocytoma cell line (PC12) cells, and in glioma cells results in activation of Jun kinase (JNK), accumulation of cytochrome c within the cytosol, and activation of caspases 9, 6, and 3. To link p75NTR-dependent JNK activation to mitochondrial cytochrome c release, regulation of BH3-domain-only family members was examined. Transcription of BH3-domain-only family members was not induced by p75NTR, but p75NTR-dependent JNK activation resulted in phosphorylation and oligomerization of the BH3-domain-only family member Bad. Loss of function experiments using Bad dominant negatives or RNA interference demonstrated a requirement for Bad in p75NTR-induced apoptosis. Together, these studies provide the first data linking apoptosis induced by p75NTR to the phosphorylation of BH3-domain-only family members.

Figure 1.

Overexpression of p75NTR reduces survival in a variety of cell types. A, Cortical neurons; B, PC12; C, U343 (wild-type p53); D, U373 (mutant p53) cells were infected with increasing multiplicities of infection (MOI) of LacZ or p75NTR recombinant adenovirus and then analyzed for survival by the MTT assay (see Materials and Methods). Error bars indicate SD. Results were analyzed for statistical significance by ANOVA (Tukey's HSD multiple comparison). Statistically significant differences of p < 0.001 are indicated by an asterisk.

Figure 2.

p75NTR activates caspases and induces accumulation of cytosolic cytochrome c. A, Cortical neurons infected with 10, 50, or 100 MOI of LacZ or p75NTR recombinant adenovirus were lysed and analyzed by immunoblot for levels of LacZ, p75NTR, and full-length caspase 9 protein or, using cleavage-specific antibodies, for levels of cleaved caspases 3 and 6 and cleaved PARP. B, U373 cells were infected with 50 or 100 MOI of either LacZ or p75NTR adenovirus for 48 hr or treated with etoposide 50 μm (+), and then lysed and analyzed for increases in cleaved caspase 9. C, E15 cortical neurons, U373, and PC12 cells were left uninfected (0) or were infected with 100 MOI of LacZ (Lz), or p75NTR (p75) recombinant adenovirus. Thirty hours later, cells were fractionated for cytosolic components as described in Materials and Methods. Cytosolic fractions normalized for protein content were analyzed by immunoblotting with an antibody directed against cytochrome c.

Figure 3.

p75NTR activates the JNK pathway. A, U373 cells were infected with 0, 50, 100, or 200 MOI of control AdLacZ or with Adp75NTR. B, PC12 cells were injected with 0 or 50 MOI of AdLacZ or Adp75NTR. C, Cortical neurons were infected with 10, 50, or 150 MOI of AdLacZ or Adp75NTR. Lysates were prepared 30-48 hr after infection and examined by immunoblot for LacZ, p75NTR, phosphorylated JNK (pJNK), total JNK (SC-474 for A, CS-9252 for B), phosphorylated c-Jun (pJun), and total c-Jun as indicated.

Figure 4.

Inhibition of MAP3K signaling attenuates apoptosis induced by p75NTR. A, U373 cells infected with 100 MOI of MLK3 adenovirus or left uninfected (0) were treated 47 hr later with DMSO or CEP1347 at 200 nm for 1 hr. Cells were harvested, and lysates were subjected to immunoblot analysis for phospho-Ser⁶³ c-Jun (pJun) and total c-Jun protein. B, Cortical neurons infected with 50 MOI of LacZ or p75NTR adenovirus were treated with DMSO or 50, 200, or 500 nm CEP1347 for 1 hr as in A. Lysates were analyzed by immunoblot as indicated (pJun, c-Jun, LacZ, p75NTR). C, AdLacZ or Adp75NTR-infected cortical neurons were treated with 500 nm CEP1347 (C) or DMSO (D) at the time of infection, and lysates were prepared 48 hr later and analyzed by immunoblot for levels of p75NTR, LacZ, phospho-Ser⁶³ c-Jun (pJun), and cleaved caspase 3 (cl. caspase 3).

Figure 5.

Activation of the JNK pathway is required for p75NTR-mediated caspase activation. Immunoblots for phospho-Ser⁶³ c-Jun (pJun), c-Jun, Flag-JIP, LacZ, p75NTR, phospho-Thr¹⁸³/Tyr¹⁸⁵-JNK (pJNK), JNK, and cleaved caspase 3 were performed as indicated on lysates from U373 cells treated with TNF 20 ng/ml that were either left uninfected (0) or infected with JBD-JIP adenovirus (JBD) at 10 MOI (A), cortical neurons infected with 50 MOI of LacZ or p75NTR adenovirus together with increasing amounts (0, 0.05, 0.5, 2.5 MOI) of JBD-JIP adenovirus (B), and PC12 cells infected with 50 MOI of LacZ or p75NTR adenovirus supplemented with LacZ or JBD-JIP (JBD) adenovirus (both at 5 MOI) (C).

Figure 6.

p75NTR-induced caspase 3 cleavage does not correlate with phosphorylation of c-Jun. PC12 cells were infected with 50 MOI of AdLacZ (Lz), Adp75NTR (p75), or AdMLK3 (MLK) recombinant adenovirus, and lysates were prepared at 30 hr after infection (A, C), or at 12, 18, 24, and 30 hr after infection (B). Lysates normalized for protein content were analyzed for LacZ, p75NTR, cleaved caspase 3, phospho-Thr¹⁸³/Tyr¹⁸⁵-JNK (pJNK), total JNK, phospho-Ser⁶³ c-Jun (pJun), and total c-Jun protein levels by immunoblot as indicated.

Figure 7.

p75NTR does not transcriptionally regulate BH3-domain-only proteins. Cortical neurons were infected with 0, 50, or 200 MOI of LacZ or p75NTR (p75) adenovirus, and 24 hr later mRNA was isolated as described in Materials and Methods. RT-PCR was performed using primers directed against Bim, Bmf, Hrk, Bik, Puma, Noxa, p75NTR, and Actin as indicated.

Figure 8.

p75NTR activates JNK-dependent phosphorylation and oligomerization of Bad. A, U373 cells were infected with 0, 50, 100, or 200 MOI of LacZ or p75NTR adenovirus, and lysates were analyzed by immunoblot for LacZ, p75NTR, phospho-Ser¹²⁸ Bad, and Bad (C-20, shown; N19, data not shown). B, PC12 cells were left uninfected (0) or were infected with LacZ (Lz) or p75NTR (p75) adenovirus aqt 100 MOI, and lysates were analyzed by immunoblot for LacZ, p75NTR, phospho-Ser¹²⁸ Bad, and Bad (C-20). C, PC12 cells were infected with nothing (0), LacZ (Lz), or p75NTR (p75) adenovirus together with either 5 MOI of LacZ or JBD-JIP (JBD) adenovirus. Lysates were compared for expression of Bad, cleaved caspase 3, LacZ, p75NTR, and Flag-JIP (Flag) by immunoblot as indicated.

Figure 9.

Bad is required for p75NTR-induced apoptosis. PC12 cells were transfected with GFP plasmid alone or with GFP plasmid together with plasmids encoding DN-Bad (S128A) or expressing Bad-RNAi. Cells were infected 48 hr later with LacZ or p75NTR adenovirus and, at 24 hr after infection, were fixed and immunostained for cleaved caspase 3 as described in Materials and Methods. Control experiments established that cleaved caspase 3 immunoreactivity correlates with TUNEL staining and is thus a valid surrogate for direct measurement of apoptosis (see supplementary Fig. 1, available at www.jneurosci.org). Transfected cells were scored for caspase 3 cleavage by a blind observer (n = 300 cells/condition). ^**Indicates a difference of p < 0.001 between GFP/Mock (Bar 1) and GFP/p75NTR (Bar 5), and ^*indicates a difference of p < 0.001 between GFP/p75NTR (Bar 5) and both DN-Bad/p75NTR (Bar 6) and with Bad RNAi/p75NTR (Bar 7), indicated by ANOVA.