|Home | About | Journals | Submit | Contact Us | Français|
NGF is recognized for its role in neuronal differentiation and maintenance. Differentiation of PC12 cells by NGF involves p53, a transcription factor that controls growth arrest and apoptosis. We investigated NGF influence over p53 activity during NO-induced apoptosis by sodium nitroprusside in differentiated and mitotic PC12 cells. NGF-differentiation produced increased p53 levels, nuclear localization and sequence-specific DNA binding. Apoptosis in mitotic cells also produced these events but the accompanying activation of caspases 1–10 and mitochondrial depolarization were inhibited during NGF differentiation and could be reversed in p53-silenced cells. Transcriptional regulation of PUMA and survivin expression were not inhibited by NGF, although NO-induced mitochondrial depolarization was dependent upon de novo gene transcription and only occurred in mitotic cells. We conclude that NGF mediates prosurvival signaling by increasing factors such as Bcl-2 and p21Waf1/Cip1 without altering p53 transcriptional activity and prevents mitochondrial depolarization and caspase activation to inhibit apoptosis.
Control over the cell death process is critical in tissues throughout the body, particularly within the nervous system. During development, apoptotic manicuring of nervous tissue is mandatory to ensure proper neuroanatomy and connectivity, as demonstrated in mice lacking key mediators of the apoptotic process . Conversely, aberrant neuronal apoptosis within the mature nervous system may play an important role in neurodegenerative diseases such as Parkinson’s, Alzheimer’s, amyotrophic lateral sclerosis and Huntington’s as well as ischemic brain conditions .
The machinery executing the apoptotic program is largely conserved throughout diverse cell types and the transcription factor p53 is recognized as a central node in apoptosis. A variety of cellular stresses transcriptionally activate p53 and its effector genes such as the cdk inhibitor, p21Waf1/Cip1, for growth arrest or apoptotic BH3 family members such as Bax and PUMA. These two cell fates are triggered by signal transduction pathways that converge on p53 to increase its nuclear localization, DNA-binding and recruitment of specific coactivators to tip the balance between expression of apoptotic and prosurvival genes . In addition, direct cooperation of cytosolic p53 with apoptotic BH3-domain proteins can contribute to mitochondrial-mediated apoptosis [4, 5]. Both apoptotic mechanisms of p53 involve the mitochondria, where permeabilization by apoptotic BH3-domain family members induce the cytosolic release of cytochrome c and initiation of the cell death program .
Recent evidence indicates the presence of p53 transcriptional activity in neuronal differentiation and neurite outgrowth in the nervous system [7, 8]. In particular, nerve growth factor (NGF) mediated differentiation of rat PC12 pheochromocytoma cells is associated with elevated p53 protein levels, increased p53 transcriptional activity and a rise in p21Waf1/Cip1 expression which is prevented by temperature sensitive mutant p53 . NGF is a neurotrophin whose signaling through the TrkA receptor  maintains neuronal differentiation in PC12 cells . NGF is believed to act as a survival factor in neuronal cells  through mechanisms that include activation of protein kinase B/Akt [13, 14] and ERK kinase through Ras activation . NGF signaling also upregulates heme oxygenase-1 [16, 17], increases glutathione synthase activity  and induces inhibitor of apoptosis (IAP) proteins [9, 19]. Consistent with its pro-survival role, NGF treatment confers some protection against a variety of cytotoxic insults including nitric oxide although the mechanism is unknown .
Nitric oxide (NO) is a diffusible diatomic nitrogen radical involved in signal transduction throughout the body  and is a contributing component to NGF-induced PC12 differentiation . Within the nervous system, NO normally functions as an endogenous neurotransmitter  but exposure to levels beyond those necessary for physiological activity may be highly toxic in neuronal cells . Low doses of NO can also be cytoprotective , mainly through a cGMP-mediated signaling mechanism [21, 25–27] or through caspase inhibition . Elevated levels of NO can activate the p53 pathway  and induce apoptosis in some cell types [30, 31]. Local immune hyperstimulation [32–34] is the major route through which neuronal cells are exposed to supraphysiological levels of reactive oxygen and nitrogen species including NO , and is thought to contribute to neurodegenerative conditions such as Parkinson’s disease . As an agent of known neurotoxicological relevance, NO represents a biologically relevant mediator of apoptosis through which cellular NGF and p53 signaling can be studied.
We hypothesized that NO-induced apoptosis involves p53 signaling in propagating PC12 cells and that NGF-induced differentiation of these cells would modulate the role of p53 during apoptosis. These studies demonstrated that NO-induced apoptosis in propagating PC12 cells was dependent on p53 activity and directly involved apoptotic gene transcription, caspase activation, depolarization of the mitochondrial membrane and nuclease cleavage of DNA. NGF-mediated differentiation was sufficient to substantially inhibit mitochondrial depolarization, caspase activation and DNA cleavage following nitroprusside treatment. However, transcriptional activity of representative p53-regulated apoptotic genes was comparable to propagating cells. We conclude that NGF mediates potent anti-apoptotic signaling by increasing factors such as Bcl-2 and p21Waf1/Cip1 and inhibiting caspase activation in neuronal PC12 cells without directly altering p53 transcriptional activity.
Rat PC12 cells (ATCC, Manassas, VA) were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% horse serum (Invitrogen), 5% fetal calf serum (Invitrogen), 4 mM L-glutamine (Invitrogen) and penicillin/streptomycin antibiotics in a humidified 37° C incubator maintained at 5% CO2. Cells of passage number 18–22 were plated on rat tail type I collagen (Sigma, St. Louis, MO) prior to experimentation and were differentiated by the addition of 50 ng/mL NGF 2.5S (Chemicon, Temecula, CA) in RPMI 1640 with 1% horse serum and antibiotics. Medium and NGF were replenished every other day until differentiation was completed at 7 days.
Sodium nitroprusside (Sigma) of 100% purity as determined by silver nitrate titration was dissolved into sterile ddH2O and added directly into culture medium at indicated concentrations for treatments. Medium was replaced prior to nitroprusside treatments in all experiments in order to maintain glucose levels and avoid widespread cellular death via necrosis . Cell number was maintained at roughly 3×104 cells per cm² of vessel surface area to ensure equivalent nitroprusside dosage per cell across experiments and culture vessel types. Cells were exposed to ultraviolet light as a positive apoptosis-inducing control at indicated doses using a Stratalinker (Stratagene, La Jolla, CA). Transcriptional inhibition was performed by incubation with 1 µM Actinomycin D (Sigma) for 1 hour prior to the indicated treatments.
Stable anti-p53 shRNA expressing PC12 cells (p53sh#3) were generated via lentiviral-mediated infection, integration and stable selection (BlockIt Lentiviral Expression System, Invitrogen). Hairpin sequences were designed and synthesized (IDT, Coralville, IA) with appropriate overhangs for cloning into the pENTR/U6 entry vector. The informative p53-targeting sense hairpin sequence is - 5′-ATATCCGACTGTGAATCCTCC-3′. shRNA was designed using the 4-base loop sequence CGAA. Hairpin sequences were cloned into the pENTR/U6 vector and selected clones were sequence-verified. The U6 promoter-shRNA cassettes were transferred into the pLenti6-BLOCK-iT-DEST vector. The resulting destination clones were used to generate lentiviral particles by transfection of HEK293FT cells along with the pLP1, pLP2 and pLP/VSVG plasmids in equal amounts using FuGene (Roche Diagnostics, Indianapolis, IN) lipid reagent. Transfection efficiencies were monitored by concomitant transfection of the pEYFP-C1 vector (BD Clontech, Mountain View, CA), demonstrating efficiency ≥ 90% in all cases. Lentiviral-laden media was collected 48 hours following transfection, clarified by centrifugation and used with 4 µg/mL polybrene (Sigma) in transduction of naïve PC12 cells plated on collagen. Selection using 6 µg/mL blasticidin was initiated and maintained for 7 days until stable lines were enriched. Stable p53shRNA-expressing cells were maintained for 14 days and passage 20–25 were used for experimentation.
Sample preparation for SDS-PAGE was performed as previously described . Cell lysate protein determination was performed using the BCA protein assay (Pierce Biotechnology, Rockford, IL). Antibodies used were against p53 (pAb122, BD Pharmingen, San Diego, CA), phospho-serine15 p53 (9284, Cell Signaling, Danvers, MA), p21Waf1/Cip1 (C-19, Santa Cruz Biotechnology, Santa Cruz, CA), Bcl-2 (DC21, Santa Cruz Biotechnology), Bcl-X(L) (2762, Cell Signaling), actin (mAb1201, Chemicon) and SULT 2A1 (Abcam, Cambridge, MA). Proteins were visualized using either ECL reagent (Amersham Biosciences, Piscataway, NJ) or SuperSignal (Pierce Biotechnology).
ChIP assays were performed similarly to previous reports [39, 40]. NGF-differentiated and naïve (dividing) PC12 cells were crosslinked by 1% formaldehyde for 15 minutes before terminating the crosslinking reaction with 125 mM glycine. Cells were washed, lysed by Dounce homogenization and resupended in a buffer containing sodium deoxycholate, SDS and Triton X-100 . Chromatin was sheared to an average length of 500 bp by sonication on ice and samples were clarified by centrifugation at 10,000xg. Sheared, crosslinked chromatin complexes were precleared with protein A agarose. p53-DNA complexes were immunoprecipitated using anti-full length p53 polyclonal antibody (FL-393, Santa Cruz Biotechnology) or with control rabbit IgG (I-5006, Sigma). Immune complexes were captured with protein A agarose and beads were washed as described [39, 40]. Protein/DNA complexes were eluted and crosslinks reversed by incubating at 65 °C for at least 6 hours. To assay for p53 binding sites in purified ChIP DNA, target specific primers were used to measure amounts of target sequence in immunoprecipitated samples by qPCR using SYBR Green-based detection (BioRad, Richmond, CA). Experimental qPCR values were normalized against values obtained for 25 ng of input DNA using the same primer set.
Cell viability was determined by measuring mitochondrial reduction of the MTS dye [3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reagent into a soluble formazan product (Promega, Madison, WI). PC12 cells were plated on collagen-coated 96-well plates and then treated as indicated for 24 hours prior to MTS addition directly to culture medium. Absorbance measurements at 490 nm were carried out within 1–2 hours using a SpectraMax M2 spectrophotometer (Molecular Devices, Sunnyvale, CA).
Caspase 3/7 activity was determined by measuring cleavage of the Z-DEVD-R110 substrate into fluorogenic product (Promega). PC12 cells were plated as above, nitroprusside-treated for 24 hours and lysed in-well along with the addition of caspase substrate. Cleavage of substrate to the fluorogenic product was measured within 2–4 hours at an excitation wavelength of 485 nm and emission wavelength of 530 nm using a SpectraMax M2 spectrophotometer as above. Pan-caspase activity was measured following described treatment in 50 µg of cellular lysates essentially as above using AFC-conjugated caspase 1–10 substrates (BioVision). Individual caspase activities were determined by measuring generation of the cleaved substrate fluorophore using excitation and emission wavelengths of 400 and 505 nm, respectively, as above.
Microscopy was performed using an Olympus IX70 inverted microscope (Olympus, Center Valley, PA). Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) was performed according to manufacturer’s instructions (Clontech, Mountain View, CA). Naïve or differentiated wild-type and anti-p53 shRNA expressing cells were plated on poly-L-lysine/collagen (Sigma) coated Lab-Tek chamber slides (Nunc, Rochester, NY) and treated as indicated. Samples were fixed in 4% formaldehyde and permeabilized in 0.2% Triton X-100 / PBS. Cleaved apoptotic DNA ends were labeled with fluorescein-dUTP by the TdT enzyme for 60 minutes at 37°C. Samples were washed and mounted using ProLong Gold reagent (Invitrogen, Carlsbad, CA) containing the nuclear counterstain DAPI and photographed under fluorescence microscopy with appropriate filter sets. Image exposure time for each fluorophore was maintained across all samples. Merging of images was performed using AxioVision software (Carl Zeiss, Oberkochen, Germany).
Mitochondria were visualized using the fixable fluorescent dye MitoTracker Red CMXRos (Invitrogen Molecular Probes, Carlsbad, CA), a cell permeable dye that is selectively sequestered in mitochondria retaining their membrane potential ΔΨ. Naïve or differentiated wild-type and shRNA expressing cells were plated on coated Lab-Tek chamber slides as above and treated as indicated. Cell populations were then exposed to 400 nM CMXRos for 10 minutes. Samples were subsequently fixed using 3.7% formaldehyde and permeabilized with 0.2% Triton X-100 / PBS for 5 minutes. Permeabilization was performed to improve CMXRos signal retention as per manufacturer’s recommended protocol. Samples were mounted using ProLong Gold antifade reagent containing DAPI, and visualized as described above with equivalent image exposure times for all samples.
Indirect immunofluorescence for p53 protein was performed on both wild-type naïve and NGF-differentiated PC12 cells plated as above. Cells were treated as indicated and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Cells were permeabilized with 0.4% Triton X-100 and incubated with FL-393 anti-p53 polyclonal antibody (Santa Cruz). p53 protein subcellular localization was labeled by incubation with goat anti-rabbit Alexa 594-conjugated secondary antibody (Invitrogen). Samples were mounted as described above and visualized using appropriate filter sets. Image exposure times were maintained across samples. Merged images were generated using AxioVision software as above.
PC12 cells and anti-p53 shRNA expressing cells were maintained in the propagating state or NGF-differentiated as above, treated and harvested at indicated intervals. Total RNA was isolated according to manufacturer’s protocol (Qiagen, Valencia, CA). RNA concentration was determined using a NanoDrop spectrophotometer (BioRad). Equal amounts of each RNA sample (1.5 µg) were used for first-strand cDNA synthesis (Invitrogen). RT-PCR was carried out using 1 µL of cDNA per sample, HotStart master mix (SuperArray, Frederick, MD) and 0.25 µM each primer (IDT) in a GeneAmp 9700 PCR instrument (Applied Biosystems, Foster City, CA). Equivalent volume of each PCR reaction was run on 2% TBE agarose gels containing ethidium bromide and photographed under UV illumination. Each target was amplified using an appropriate, empirically determined cycle number allowing gel-based visualization of samples within the exponential amplification phase of each reaction. Primer sequences are as follows: p53 forward 5’-CAGCCAAGTCTGTTATGTGC-3’, p53 reverse 5’-GTCTTCCAGCGTGATGATG-3’, p21Waf1/Cip1 forward 5’-TCCTTGCCACTTCTTACCTG-3’, p21Waf1/Cip1 reverse 5’-CCGGGCATCTTTGTTCTAG-3’, MDM2 forward 5’-GTGACCATTCTGCTGATTGC-3’, MDM2 reverse 5’-CGCTTTCTCCTGCCTGATAG-3’, cyclin G1 forward 5’-ATTGCTGCCTCAATCTAGTC-3’, cyclin G1 reverse 5’-CCTGGAGTGTTTTATCAAGC-3’, PUMA forward 5’-CCTGCCTCACCTTCATCTG-3’, PUMA reverse 5’-TCCCTGACTCCCCATCTTC-3’, survivin forward 5’-GGAGACCAACAACAAACAG-3’, survivin reverse 5’-CAGGTCACAATAGAGCAAAG-3’, Bad forward 5’-CAACACAGATGCGACAAAG-3’, Bad reverse 5’-AACGGAGAGGGAACATAGC-3’, Bid forward 5’-GGAGGAAGACAAAAGGAAC-3’, Bid reverse 5’-AGCCGTAAGACCAAGGTAC-3’, Bax forward 5’-GCGTGGTTGCCCTCTTCTAC-3’, Bax reverse 5’-CAGTGTCCAGCCCATGATG-3’, GAPDH forward 5’-ATCCCATCACCATCTTCCAG-3’, GAPDH reverse 5’-CCTGCTTCACCACCTTCTTG-3’.
Group comparison of MTS viability assay data (e.g. naïve cells across range of nitroprusside doses) was performed using one-way ANOVA (p≤0.05) followed by Tukey’s Honest Significant Difference post-hoc test. Comparison of viability between naïve and differentiated cells at individual nitroprusside doses were performed by two-tailed Students’ t-test, where data were statistically significant at p≤0.05. Chromatin immunoprecipitation enrichment was compared between naïve and NGF-differentiated cells at each genomic locus using two-tailed t-test with significance at p≤0.05. Caspase activity significance was calculated using two-tailed t-test at p≤0.05.
Cell viability was measured in both naïve and NGF-differentiated PC12 cells following treatment with increasing concentrations of nitroprusside in both naïve and NGF-differentiated PC12 cells using the MTS reagent (Figure 1). We demonstrate that nitroprusside exposure reduces cell viability in both naïve and NGF-differentiated PC12 cells in a concentration-dependent manner, with differentiated cells significantly less susceptible to loss of cell viability at higher concentrations. At doses of 0.8 mM and 1 mM nitroprusside, 22% and 15.21% more mitotic (naïve) cells, respectively, were nonviable at these concentrations compared to differentiated cells.
p53 protein was found to accumulate in both a concentration- and time-dependent manner following nitroprusside treatment in naïve PC12 cells (Figures 2A, 2B), with levels proportionately elevated following doses of 0.3 mM through 1 mM over the course of 24 hours. Accumulated p53 protein was posttranslationally phosphorylated at the serine 15 residue in a dose-dependent manner, consistent with p53 transcriptional activation  in these cells (Figure 2A). p53 protein levels were quickly elevated over time in naïve cells following 0.8 mM nitroprusside, with levels of p53 protein visible within 30 minutes (Figure 2B). p53 phosphorylation at serine 15 occurred in a delayed manner compared to levels of total p53 protein (Figure 2B) which is typically observed during p53 activation.
p53 protein levels were highly elevated in response to NGF treatment alone in PC12 cells over the course of 7 days (Figure 2C). Elevated p53 protein levels were accompanied by NGF-dependent increases in DNA binding to regulatory elements of the known target genes p21Waf1/Cip1 and MDM2 (Figure 2D) as determined via chromatin immunoprecipitation (ChIP) assay. Binding to the proximal p53 binding site (relative to the +1 transcriptional start site) within the p21Waf1/Cip1 promoter was increased 3.3-fold following NGF treatment, while binding to the distal p53 response element was elevated 3.9-fold. Occupancy of the intronic primary p53 binding site within the MDM2 gene was also elevated in response to NGF alone, increasing by 3.7-fold compared to naïve cells. Following nitroprusside treatment of differentiated cells, p53 protein levels were moderately decreased at 2 hours and quickly returned to the highly elevated levels observed in unstressed NGF-differentiated cells within 4 hours (Figure 2E).
Compared to untreated controls, we observed both an elevation in p53 levels and an increase in nuclear localization in nitroprusside-treated naïve PC12 cells within 4 hours (Figure 2F). Untreated NGF-differentiated cells contained prominent nuclear p53 protein as determined by both immunofluorescence and subcellular fractionation coupled with immunoblotting (data not shown), while nitroprusside exposure further increased p53 nuclear localization within 4 hours of treatment.
Both naïve and differentiated PC12 cells were treated with 0.8 mM nitroprusside. Pan-caspase activity (caspases 1–10) was measured at 18 hours, the time point following treatment at which the greatest fluorescence was generated based on preliminary experiments. Caspase activity in naïve PC12 cells was significantly activated within 18 hours following nitroprusside treatment (Figure 3A). Caspase 3 showed the most significant activation resulting in a 2.3-fold activity increase relative to untreated naïve cells. In contrast, NGF-differentiated cells demonstrated insignificant changes in the activities of caspases 1–10 following nitroprusside treatment over the course of 18 hours (Figure 3B).
We questioned whether attenuated caspase activity in differentiated PC12 cells was dose-dependent relative to naïve caspase activity. As expected, naïve PC12 cells underwent marked caspase 3 activation across a range of nitroprusside doses (Figure 3C), with a 2.2-fold increase in activity following 0.8 mM nitroprusside, consistent with previous caspase activity data. Caspase 3 activity was also elevated at lower concentrations (0.1 through 0.5 mM) of nitroprusside in naïve cells although significant levels of apoptosis were not yet visually observed. In contrast, no tested concentration of nitroprusside elicited an increase in caspase 3 activity within differentiated cells (Figure 3D). The greater reductions in caspase 3 activity in both naïve (Fig 3C) and differentiated cells (Fig 3D) observed as nitroprusside doses approached 1mM, when coupled with the observation that similar doses of nitroprusside lead to increasing cell death (Fig 1), suggest that high levels of nitroprusside also leads to acute necrosis irrespective of NGF signaling.
PC12 cell lines were generated which stably express anti-p53 shRNA from a constitutively active U6 promoter to maintain lower cellular levels of p53 RNA and protein compared to their wild-type counterparts. Levels of p53 RNA in shRNA-expressing p53sh#3 cells were significantly reduced compared to both the naïve and differentiated wild-type PC12 cells (Figure 4A). Stable silencing of p53 RNA efficiently maintained low p53 protein levels as determined by p53 immunoblot even in the presence of NGF (Figure 4B), which was observed earlier (Figure 2C) to effectively elevate p53 protein levels in PC12 cells.
We hypothesized that if nitroprusside-induced caspase activation produced apoptosis by a p53-dependent mechanism then reduction of p53 should limit apoptosis in PC12 cells. Apoptosis was measured in both wild-type and p53-silenced naïve and differentiated PC12 cells by TUNEL assay (TdT biotin-dUTP Nick End Labeling). Naïve wild-type PC12 cells undergo marked apoptosis after nitroprusside exposure as determined by the proportion of fluorescent TUNEL-positive cells compared to untreated control (Figure 4C). Conversely, the number of apoptotic TUNEL-positive cells were substantially reduced in naïve p53-silenced p53sh#3 cells. NGF-differentiated cells were substantially more resistant to nitroprusside-induced apoptosis when compared to naïve cells (Figure 4D). However, a small number of differentiated cells were observed as TUNEL-positive after nitroprusside which was similar and not further reduced in differentiated p53sh#3 cells.
Nitric oxide is known to produce cell death through mixed necrosis and apoptosis in some cell types [37, 42]. Using the stain trypan blue, we scored cells for membrane permeability as an indicator of necrosis within naïve and NGF-differentiated cells following 20 hours 0.8 mM nitroprusside exposure. In naïve cells, nitroprusside treatment produced 17.2% ± 3.4% trypan blue-positive cells (n=3) and 11.9% ± 0.64% trypan blue-positive cells in NGF-differentiated PC12 cells (n=3). These data suggest that levels of necrosis following nitroprusside treatment were similar in naïve and differentiated cells.
Maintenance of the mitochondrial membrane potential ΔΨ following nitroprusside treatment was studied in both naïve and differentiated PC12 cells using the cell permeant dye, MitoRed CMXRos, which accumulates selectively within mitochondria maintaining a potential difference across their membrane. Naïve PC12 cells exposed to 0.8 mM nitroprusside experienced marked loss of mitochondrial membrane potential compared to untreated cells (Figure 5A), as evidenced by decreased mitochondrial MitoRed dye retention. Loss of mitochondrial membrane potential in naïve cells was concurrent with pyknotic nuclei in DAPI counterstained naïve cells suggesting a positive relationship between apoptosis and mitochondrial pathology. Rare large nuclei consistent with necrotic pathology were also observed, both with and without MitoRed staining, in nitroprusside-treated naïve cells although less frequently observed in NGF-differentiated cells. Mitochondrial depolarization was largely inhibited in p53-silenced p53sh#3 naïve cells following nitroprusside treatment. Furthermore, nuclear changes consistent with apoptosis were visibly absent in nitroprusside-treated naïve p53sh#3 cells, suggesting that p53 was involved in both mitochondrial and nuclear pathology following nitroprusside treatment in the naïve cell. Differentiated PC12 cells were substantially less susceptible to nitroprusside-mediated loss of mitochondrial membrane potential (Figure 5B). Both wild-type and p53sh#3 cells treated with NGF retained MitoRed fluorescence following nitroprusside exposure, in contrast to naïve PC12 cells.
Next we examined the pathways for nitroprusside-induced apoptosis in naïve PC12 cells for comparison to NGF-differentiated cells. Although rapid p53 nuclear partitioning was demonstrated in response to nitroprusside exposure (Figure 2F), we attempted to rule out proapoptotic functions of cytoplasmic p53 [4, 5, 43], as a mechanism leading to nitroprusside toxicity.
Cellular RNA synthesis was inhibited by pretreatment with 1 µM Actinomycin D one hour prior to nitroprusside treatment to determine if p53 transcriptional function was upstream of mitochondrial depolarization. No apparent nuclear or mitochondrial pathology was detected following treatment with Actinomycin D alone (Figure 6A). As expected in naïve PC12 cells, nuclear pyknosis and mitochondrial depolarization were apparent following nitroprusside treatment (Figure 6A). In combination with nitroprusside, Actinomycin D significantly alleviated, although did not completely prevent, chromatin pyknosis and mitochondrial depolarization in naïve cells.
Both p53 silencing and generalized transcriptional inhibition by Actinomycin D were protective against nitroprusside-induced mitochondrial pathology in naïve PC12 cells. Therefore, it was likely that p53-dependent transcriptional regulation of apoptotic target genes controlled the response to nitroprusside treatment. We hypothesized that if NGF inhibited apoptosis by directly modulating p53 activity then p53-dependent transactivation or repression of target genes would be affected. Using both wild type and p53sh#3 cells, we found that gene expression of the known target genes p21Waf1/Cip1, MDM2 and cyclin G1 were increased in both naïve and NGF-differentiated cells following nitroprusside treatment in a p53-dependent manner (Figure 6B). As expected by earlier ChIP analyses in Figure 2D, the transcript levels of p21Waf1/Cip1 and MDM2 were highly elevated in NGF-differentiated compared to naïve cells during nitroprusside toxicity. We observed that expression of the apoptotic gene, PUMA, was highly increased in a p53-dependent manner with nitroprusside in NGF-differentiated cells, but only moderately increased in the naïve cell. Anti-apoptotic survivin expression was also repressed following nitroprusside in all cell types. Although survivin is a recognized target of p53 in apoptosis [44, 45], this gene may also be regulated by additional mechanisms following p53 suppression. mRNA levels of the apoptotic BH3-domain protein, Bad, were increased slightly at 4 hours following nitroprusside treatment in differentiated cells, while no changes were observed in expression levels of the apoptotic BH3-domain family members, Bid or Bax, in either the naïve or NGF-differentiated state following nitroprusside treatment. These results demonstrated that transcription of p53 targets involved in both apoptosis and cell cycle arrest were highly similar in both the naïve and NGF-differentiated cells treated with nitroprusside.
As a potential mechanism of NGF-induced protection against apoptosis, levels of the anti-apoptotic proteins Bcl-2 and Bcl-X(L) were studied during PC12 differentiation. We observed that Bcl-2, but not Bcl-X(L), protein levels were increased over the course of 7 days during NGF differentiation (Figure 7A). Upon nitroprusside treatment, anti-apoptotic Bcl-2 protein levels in naïve cells decreased within 7 hours while expression in NGF-differentiated cells remained constant (Figure 7B), in agreement with observed levels of apoptosis (Figure 4C and 4D). In contrast, p21Waf1/Cip1 protein levels increased over time in naïve cells upon nitroprusside treatment, with greatest amounts seen within 12–24 hours (Figure 7C). In accordance with p53 occupancy and mRNA expression data (Figure 2D and and6B,6B, respectively), p21Waf1/Cip1 levels were highly elevated in the untreated differentiated cell and remained elevated with no change in p21Waf1/Cip1 expression observed after nitroprusside exposure (Figure 7C).
These studies demonstrated that NGF inhibited p53-dependent apoptosis induced by supraphysiological levels of NO after nitroprusside treatment. NGF-mediated inhibition of apoptotic p53 signaling is particularly interesting because NGF itself strongly induces the nuclear accumulation and transcriptional activity of p53 during neuronal PC12 differentiation. As described here, NGF-mediated activation of p53 within the differentiating PC12 cell does not result in apoptosis, suggesting the presence of an NGF-dependent mechanism through which apoptotic p53 activity was suppressed. We observed potent NGF-mediated inhibition of both caspase activation and the p53-dependent processes of mitochondrial pathology and apoptosis following nitroprusside exposure. NGF-induced differentiation results in increased PC12 cellular surface area, suggesting that differentiated cells may be exposed to doses of nitroprusside proportionately higher than their naïve counterparts. Because NGF-differentiated cells are significantly protected from nitroprusside-induced apoptosis, this exposure model further reflects the pro-survival effect of NGF signaling in PC12 cells. Furthermore, NGF-regulated pathways promoted neuronal cell survival during differentiation even though p53-dependent transcription of apoptotic effector proteins was increased. We therefore conclude that NGF anti-apoptotic activity was not mediated by the direct alteration of p53 transcriptional activity.
Expression of factors affecting mitochondrial integrity in the differentiation process may be important for cell survival. Mitochondrial depolarization has been shown to contribute to apoptosis in various cell types [46–49], including nitroprusside-treated PC12 cells . However, the role of NGF and p53 in nitroprusside-induced mitochondrial depolarization is not known. NGF treatment enhanced PC12 cell survival during nitroprusside exposure despite concurrent expression of apoptotic p53 target genes PUMA and Bad, and over the repression of anti-apoptotic survivin expression. NGF alone increased Bcl-2 expression as previously described , which might account for some of NGF’s pro-survival effects in PC12 cells although the exact role for Bcl-2 in preventing mitochondrial depolarization remains under study [52, 53]. An alternative mechanism through which apoptosis may be inhibited in the NGF-differentiated cell was by the anti-apoptotic activity of the p53 target gene p21Waf1/Cip1. The cyclin-dependent kinase inhibitor p21Waf1/Cip1 has long been recognized as the major mechanism through which p53 regulates G1 cell cycle arrest . Recent findings also suggest that p21Waf1/Cip1 acts in a dominant manner to inhibit apoptotic signaling , while loss of p21Waf1/Cip1 sensitizes cells to apoptosis . As shown here, NGF-activated p53 binds to the p21Waf1/Cip1 promoter and activates its transcription. Furthermore, p21Waf1/Cip1 protein levels were highly increased with NGF alone as previously described [9, 57]. NGF may therefore prime PC12 cells for survival by increasing levels of the antiapoptotic factors p21Waf1/Cip1 and Bcl-2 during differentiation.
NO signaling occurs through a series of reactions including nitrosylation of the guanylate cyclase heme moiety , cysteine thiol S-nitrosylation  and tyrosine nitrosation . The outcome of NO-mediated signaling may therefore be dependent upon both target availability and local NO concentration. A multitude of effects accompany NO-mediated signaling, including the inhibition of caspases at low NO levels . In this study, levels of NO from nitroprusside exposure acted as an apoptotic stimulus in naïve cells consistent with an observed rise in caspase activities. We report that activities of multiple caspases were reduced following nitroprusside treatment in differentiated but not naïve PC12 cells. Reduced caspase activity may be the result of NGF anti-apoptotic activity rather than direct NO-mediated caspase inhibition. In addition, induction of heme oxygenase-1 has been shown to protect against nitrosative stress  through decreased cytotoxicity . Our studies demonstrated that p53-regulated apoptotic gene expression upon nitroprusside exposure was enhanced in NGF-differentiated cells, suggesting that the cytotoxic insult generated by NO was similar in differentiated cells and mitotic cells. NGF is also known to activate Akt/PKB signaling in differentiating PC12 cells . While Akt/PKB is necessary for the trophic effects of NGF [64, 65], the role of the Akt/PKB pathway in pro-survival signaling elicited by NGF continues to be studied [66, 67] and may be dependent upon the apoptotic stimulus. Akt/PKB survival signaling may be carried out by increasing Bcl-2 levels during NGF-mediated differentiation . We therefore concluded that NGF-mediated inhibition of apoptosis occurs upstream of mitochondrial pathology upon nitroprusside treatment, and may include multiple protective mechanisms described above.
Changes in neurotrophin levels and their receptors within the nervous system has been suggested in neurodegenerative disorders such as Alzheimer’s  and Parkinson’s disease . Because of their protective effect within the nervous system, neurotrophin replacement has been studied as a therapeutic for spinal cord injury and excitotoxicity [71, 72] and has been suggested as a therapeutic intervention for neurodegenerative conditions . The studies described here suggest that the neurotrophin NGF has the ability to protect neuronal cells from an apoptotic stimulus in vitro, and supports the idea of NGF supplementation as a protective in vivo therapeutic agent. However, our studies demonstrated that while NGF may antagonize apoptotic stimuli in PC12 cells, it did not protect against necrotic cell death. Therefore, NGF supplementation therapy may be of limited value as a broad-spectrum protective agent against cell death in vivo.
In vitro exposures examining cytotoxicity of endogenous compounds like NO are not always applicable to complex tissue responses in vivo such as the central and peripheral nervous system where neurotrophic factors exert powerful receptor-mediated signaling . Cell cycle-dependent effects in immortalized neuronal cell models may significantly alter the toxic response compared to post-mitotic neuronal cells in culture [75, 76]. While NGF-differentiated neuronal cultures may imperfectly model the complexity of NO-mediated events in vivo, they point to the importance of using post-mitotic cells for study of neuronal cell death and neurodegeneration in vitro.
This work was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. We thank Dr. Alexandra Heinloth and Dr. Sonnet Arlander for critical review of this manuscript.