Growth arrest of PC12 cells by nerve growth factor is dependent on the phosphatidylinositol 3-kinase/Akt pathway via p75 neurotrophin receptor

We recently isolated mutant PC12 cell clones (PC84 cells) by transfection of PC12 cells with nerve growth factor (NGF) cDNA. These cells secreted active NGF and extended short processes, but proliferated faster than the parental PC12 cells. Because the expression level of p75, a low-affinity receptor for NGF, was significantly low, we suspected that NGF signaling via p75 was necessary for the growth arrest of the PC12 cells, and this was shown to be the case by repressing p75 function in PC12 cells. In this study, we examined the downstream signaling of p75, which would ultimately evoke the growth arrest. NGF is known to induce rapid phosphorylation of MAP kinase and Akt in PC12 cells, whereas in PC84 cells, MAP kinase was phosphorylated but the phosphorylation level of Akt was very low under the serum-free condition. This finding suggested that the low expression level of p75 in PC84 cells was the reason for the low Akt activation. Because Akt is known to be activated via phosphatidylinositol (PI) 3-kinase, we treated PC12 cells with a PI3-kinase inhibitor, Wortmannin, and found these cells did not cease proliferation in the presence of NGF. Furthermore, anti-p75 neutralizing antibody reduced NGF-induced phosphorylation of Akt in PC12 cells under the serum-free condition. Because we had already shown that PC12 cells treated with anti-p75 neutralizing antibody did not cease proliferation in the presence of NGF, these results suggest that NGF activates Akt via p75, which is necessary for the NGF-induced growth arrest of PC12 cells.     

Comparison of nerve growth factor receptor binding models using heterodimeric muteins

Nerve growth factor (NGF) is a homodimer that binds to two distinct receptor types, TrkA and p75, to support survival and differentiation of neurons. The high‐affinity binding on the cell surface is believed to involve a heteroreceptor complex, but its exact nature is unclear. We developed a heterodimer (heteromutein) of two NGF muteins that can bind p75 and TrkA on opposite sides of the heterodimer, but not two TrkA receptors. Previously described muteins are Δ9/13 that is TrkA negative and 7‐84‐103 that is signal selective through TrkA. The heteromutein (Htm1) was used to study the heteroreceptor complex formation and function, in the putative absence of NGF‐induced TrkA dimerization. Cellular binding assays indicated that Htm1 does not bind TrkA as efficiently as wild‐type (wt) NGF but has better affinity than either homodimeric mutein. Htm1, 7‐84‐103, and Δ9/13 were each able to compete for cold‐temperature, cold‐chase stable binding on PC12 cells, indicating that binding to p75 was required for a portion of this high‐affinity binding. Survival, neurite outgrowth, and MAPK signaling in PC12 cells also showed a reduced response for Htm1, compared with wtNGF, but was better than the parent muteins in the order wtNGF > Htm1 > 7‐84‐103 >> Δ9/13. Htm1 and 7‐84‐103 demonstrated similar levels of survival on cells expressing only TrkA. In the longstanding debate on the NGF receptor binding mechanism, our data support the ligand passing of NGF from p75 to TrkA involving a transient heteroreceptor complex of p75‐NGF‐TrkA. © 2012 Wiley Periodicals, Inc.     

NGF ligand alters NGF signaling via p75(NTR) and trkA

Nerve growth factor (NGF) binds to two neurotrophin receptors: p75(NTR) and p140(Trk) (TrkA). Both receptors dimerize in response to NGF binding. TrkA homodimers and heteromers of TrkA and p75(NTR) promote cell survival whereas homodimers of p75(NTR) mediate apoptosis upon binding of NGF. The interaction between receptor and NGF can be inhibited either on the level of the ligand by altering NGF conformation so that NGF is no longer recognized by the receptor or on the level of the receptor by blocking the binding site of p75(NTR) or TrkA. The effect of altering NGF conformation on NGF signaling was investigated in two neuron-like cell lines: in human SK-N-MC cells that express only p75(NTR) and in rat PC12 cells that express both p75(NTR) and TrkA. In the present study we demonstrate that Ro 08-2750 binds to the NGF dimer thereby probably inducing a change in its conformation such that NGF cannot bind to p75(NTR) anymore. In SK-N-MC cells this leads to inhibition of NGF-induced programmed cell death. In PC12 cells enhanced signaling through TrkA was observed.     

Differential endocytic sorting of p75NTR and TrkA in response to NGF: a role for late endosomes in TrkA trafficking

NGF binds to two receptors, p75NTR and TrkA. The endosomal trafficking of receptors is of emerging importance for the understanding of their signaling. We compared the endocytic trafficking of the two NGF receptors in PC12 cells. Both p75NTR and TrkA were internalized in response to NGF and colocalized with early endosomes. However, surprisingly, the subsequent endosomal trafficking paths of both NGF receptors diverged: whereas p75NTR recycled back to the surface, TrkA moved to late endosomes and underwent lysosomal degradation. By performing subcellular fractionations of NGF stimulated PC12 cells, tyrosine-phosphorylated TrkA was recovered in fractions corresponding to late endosomes. This implicates these organelles as novel endosomal NGF signaling platforms. Furthermore, the trafficking of NGF receptors could be manipulated by pharmacological means. Disrupting p75NTR recycling diminished TrkA activation in response to low concentrations of NGF, demonstrating a functional role for the recycling of p75NTR.     

The common neurotrophin receptor p75NTR enhances the ability of PC12 cells to resist oxidative stress by a trkA-dependent mechanism

Functional role(s) for the common neurotrophin receptor p75NTR in nerve growth factor (NGF) signaling have yet to be fully elucidated. Many studies have demonstrated that p75NTR can enhance nerve growth factor-induced survival mediated via the trkA receptor. In addition, newly identified pathways for p75NTR signaling have included distinct p75NTR-specific and trk-independent effects which generally appear to be pro-apoptotic. In the present study, we have examined the influence of p75NTR on NGF-mediated protective effects from hydrogen peroxide (H2O2)-induced apoptotic cell death of PC12 cells. Exposure of PC12 cells to H2O2 resulted in Caspase-3 activation and apoptosis. NGF protected PC12 cells against H2O2-mediated apoptosis in a dose-dependent manner and inhibited Caspase-3 activation. These effects of NGF required activation of both PI 3-kinase and MAP kinase signal pathways. When NGF binding to p75NTR was blocked by treating cells with either BDNF or PD90780, and where p75NTR expression was reduced by treating cells with antisense oligonucleotide to p75NTR, the protective effects of NGF were attenuated. Further, NGF had no effect on cell viability in PC12nn5 cells, which express only p75NTR. When trk-mediated signal transduction was blocked, leaving p75NTR signaling activated, PC12 cells were not more vulnerable to H2O2. These data suggest that p75NTR enhances the ability of PC12 cells to resist oxidative stress by a trkA-dependent mechanism, potentially by allosteric mechanisms. Further, potential trkA-independent and pro-apoptotic signaling of p75NTR does not contribute to apoptotic cell death of PC12 cells in a setting of oxidative insult.     

Reciprocal modulation of TrkA and p75NTR affinity states is mediated by direct receptor interactions

Equilibrium binding of ¹²⁵I-nerve growth factor (¹²⁵I-NGF) to cells coexpressing the tyrosine kinase receptor A (TrkA) and common neurotrophin receptor (p75^NTR), cells coexpressing both receptors where p75^NTR is occupied, and cells expressing only p75^NTR, revealed reciprocal modulation of receptor affinity states. Analysis of receptor affinity states in PC12 cells, PC12 cells in the presence of brain-derived neurotrophic factor (BDNF), and PC12^nnr5 cells suggested that liganded and unliganded p75^NTR induce a higher affinity state within TrkA, while TrkA induces a lower affinity state within p75^NTR. These data are consistent with receptor allosterism, and prompted a search for TrkA/p75^NTR complexes in the absence of NGF. Chemical crosslinking studies revealed high molecular weight receptor complexes that specifically bound ¹²⁵I-NGF, and were immunoprecipitated by antibodies to both receptors. The heteroreceptor complex of TrkA and p75^NTR alters conformation and/or dissociates in the presence of NGF, as indicated by the ability of low concentrations of NGF to prevent heteroreceptor crosslinking. These data suggest a new model of receptor interaction, whereby structural changes within a heteroreceptor complex are induced by ligand binding.     

Nerve growth factor-induced protein kinase C stimulation contributes to TrkA-dependent inhibition of p75 neurotrophin receptor sphingolipid signaling

Previous studies have established that reciprocal interactions between the low-affinity p75 nerve growth factor (NGF) receptor (p75(NTR)) and the high-affinity TrkA NGF receptor can dictate the cellular response to NGF. As the most important interaction, TrkA signaling was found to inhibit p75(NTR)-mediated sphingomyelinase (SMase) stimulation, ceramide production, and apoptosis. However, the mechanism by which TrkA counteracts p75(NTR)-coupled sphingolipid signaling is still unclear. Considering the stimulatory effect of NGF on protein kinase C (PKC) activity, we investigated the role of PKC in TrkA/p75(NTR) signaling interaction. In this study, we found that, in SK-N-BE cells, which selectively express p75(NTR), phorbol ester-induced PKC stimulation resulted in the abrogation of SMase stimulation and ceramide production induced by NGF. Moreover, in SK-N-BE neuroblastoma cells, which selectively express TrkA, NGF stimulated global PKC activity through two independent pathways involving phospholipase Cgamma (PLCgamma) and phosphoinositide-3 kinase (PI3K). In SH-SY5Y, another neuroblastoma cell line, which coexpresses TrkA and p75(NTR), NGF induced PKC stimulation through a TrkA/PI3K signaling pathway, whereas there was no ceramide production. However, in these cells, the inhibition of TrkA, PI3K, and PKC resulted in the restoration of NGF-induced ceramide production. Thus, our study demonstrates for the first time that TrkA interferes with p75(NTR) signaling through a PI3K/PKC-dependent mechanism.     

Loss of cell adhesion impairs the early response of TrkA to nerve growth factor (NGF)

Within the in vitro model of nerve growth factor (NGF)-dependent differentiation of PC12 cells, loss of adhesion is known to interfere with certain neurotrophic responses and not with others. The present analysis of early NGF signaling shows that the upstream activation of TrkA, as well as the recruitment of the adaptor protein Shc, are dramatically attenuated upon loss of cell adhesion, while the downstream activation of ERK1/2 is unaffected. That loss of cell adhesion interferes with the primary signaling response to NGF (i.e. autophosphorylation of TrkA) may explain why suspended PC12 cells fail to morphologically differentiate in response to NGF. The possibility that established adhesion-driven mechanisms underlie the full responsiveness of TrkA to NGF is discussed.     

Differential actions of nerve growth factor receptors TrkA and p75NTR in a rat model of epileptogenesis

Kindling, an experimental model of epileptogenesis, and activation-induced synaptic reorganization are modulated by nerve growth factor (NGF), but whether NGF acts via its high-affinity receptor TrkA and/or the common neurotrophin receptor p75NTR is unknown. We previously demonstrated, and confirmed in this study, that inhibition of NGF binding to both TrkA and p75NTR inhibited kindling and decreased kindling-induced mossy fiber sprouting. We now report specific inhibition of TrkA.NGF binding, but not p75NTR.NGF binding, retarded perforant path kindling progression. However, mossy fiber sprouting was inhibited by either selective TrkA.NGF or p75NTR.NGF antagonists. Our results suggest that TrkA, but not p75NTR, plays a role in kindling, while both receptors modulate kindling-induced mossy fiber sprouting. This implicates different mechanisms of neurotrophin action on kindling (mediated by TrkA) and neuronal sprouting (mediated by both TrkA and p75NTR) and suggests that sprouting involves kindling-independent neurotrophin action via p75NTR.     

Inhibition of nerve growth factor signaling by peroxynitrite

The reactive oxygen species peroxynitrite has been implicated in mediating oxidative damage within the brain, and in particular in those regions associated with the pathology of Alzheimer disease. Evidence for peroxynitrite damage includes the abundance of nitrated tyrosine residues within proteins of neural cells. Potential sites for peroxynitrite-induced cytotoxicity are the tyrosine residues of tyrosine kinase receptors that are crucial for the maintenance of cholinergic neurons. The peroxynitrite generator 3-morpholinosydnonmine (SIN-1) was used to examine the effects of peroxynitrite generation on nerve growth factor (NGF)/TrkA signaling in PC12 pheochromocytoma cells that express a cholinergic phenotype. NGF produced a concentration-dependent increase in PC12 cellular metabolism (EC(50) = 15.2 ng/ml) measured in a microphysiometer. This action of NGF was inhibited in a concentration-dependent manner up to 67% of control by a brief (20 min) exposure of the cells to SIN-1. This inhibition of the NGF cellular response by SIN-1 was not related to generalized cellular toxicity. In fact, the peroxynitrite scavenger uric acid significantly attenuated the inhibitory actions of SIN-1. Pretreatment with SIN-1 also resulted in a decrease in the NGF-induced phosphorylation of TrkA protein. Furthermore, SIN-1 treatment reduced the activity of mitogen activated protein kinase (MAPK), a downstream kinase activated by TrkA receptor stimulation. These data suggest that SIN-1 treatment inhibits NGF signaling by inactivating TrkA receptors through the formation of nitrotyrosine residues on the receptor. The inactivation of TrkA receptors may contribute to the initial insult that eventually leads to neuronal cell death.     

Nerve growth factor regulates substance P in adult sensory neurons through both TrkA and p75 receptors

Expression of the nociceptive peptide, substance P (SP) is regulated by the neurotrophin, nerve growth factor (NGF), and exogenous exposure to high levels of NGF increases its cellular content and release. NGF utilizes two receptors, the NGF-specific tyrosine kinase receptor, TrkA, and also the non-specific neurotrophin receptor, p75(NTR) (p75). The purpose of this study is to determine the relative involvement of these receptors in nociception. To investigate the role of TrkA in SP signaling, sensory neurons from adult rats were grown in vitro and exposed to a TrkA-blocking antibody. Pretreatment with the antibody inhibited NGF-induced SP elevation. Furthermore, when neurons were exposed to K252a, a relatively specific TrkA kinase inhibitor, the NGF effect on SP was also inhibited. K252a did not prevent SP up-regulation in cells exposed to forskolin or glial cell line-derived neurotrophic factor (GDNF), two agents which increase SP expression independently of TrkA. When p75 was blocked by antiserum, SP up-regulation by NGF was also inhibited. The antiserum neither impacted neuronal survival or basal levels of SP expression, nor did it inhibit SP up-regulation induced by forskolin. Two other neurotrophins, which are also ligands for p75, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) did not block NGF-induced SP up-regulation, raising the possibility that activated p75 is able to cooperate in SP regulation regardless of which neurotrophin ligand occupies it. Our data suggest that NGF up-regulation of SP expression requires the involvement of both TrkA and p75, although the specific contribution of each receptor to SP signaling remains to be determined.     

Duration and magnitude of nerve growth factor signaling depend on the ratio of p75LNTR to TrkA

The role of the low affinity neurotrophin receptor p75^LNTR in neurotrophin signal transduction remains open. Recent reports show that this receptor generates intracellular signals independent of Trk activity, and others imply that it collaborates with Trk(s) to enhance cellular responses to low neurotrophin concentrations. We have used the Cytosensor microphysiometer as a direct marker of intracellular metabolic activity to address the physiologic role of p75^LNTR in nerve growth factor (NGF) signal transduction. NGF treatment of PC12 or TrkA-transfected Chinese hamster ovary (CHO) cells results in a rapid, transient increase in the extracellular acidification rate as measured by the Cytosensor; in both cell types, p75^LNTR enhances this response. p75^LNTR affects both the magnitude of and the duration of the extracellular acidification response to NGF. Moreover, it is not merely the presence of p75^LNTR, but also the ratio of p75^LNTR:TrkA which determines cellular responsiveness to NGF. In transiently transfected CHO cells, a 5:1 ratio of p75^LNTR:trkA cDNAs produced the greatest change in NGF-induced acid secretion. Pretreatment of PC12 cells with anti-p75^LNTR antibodies decreased the responsiveness to NGF. However, long-term NGF exposure to PC12 cells in which p75^LNTR expression was decreased to approximately 10% of wild-type levels showed a longer duration of acid secretion compared to wild-type PC12 cells. Together, these data suggest that p75^LNTR may play a dual role in modulating NGF signal transduction by enhancing and extending cellular responses to short-term ligand exposures while attenuating the metabolic response to long-term ligand exposures. With regard to potential Trk-independent p75^LNTR signal transduction mechanisms, we detected no change in extracellular acidification response in 75^LNTR-transfected CHO cells, PCNA-15 fibroblasts, or Schwann cells, all of which express large amounts of p75^LNTR and no Trk. Thus, p75^LNTR cannot produce any signal detected by microphysiometry in the absence of TrkA. J. Neurosci. Res. 51:442–453, 1998. © 1998 Wiley-Liss, Inc.     

Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells

We have characterized some of the nerve growth factor (NGF) stimulated receptor tyrosine kinase (TrkA) signalling cascades in adult rat primary dorsal root ganglia (DRG) neuronal cultures and compared the pathways with those found in PC12 cells. TrkA receptors were phosphorylated on tyrosine residues in response to NGF in DRG neuronal cultures. We also saw phosphorylation of phospholipase Cγ1 (PLCγ1). We used recombinant glutathione-S-transferase (GST)–PLCγ1 SH2 domain fusion proteins to study the site of interaction of TrkA receptors with PLCγ1. TrkA receptors derived from DRG neuronal cultures bound preferentially to the amino terminal Src homology-2 (SH2) domain of PLCγ1, but there was enhanced binding with tandemly expressed amino- and carboxy-terminal SH2 domains. The most significant difference in NGF signalling between PC12 cells and DRG was with the Shc family of adapter proteins. Both ShcA and ShcC were expressed in DRG neurons but only ShcA was detected in PC12 cells. Different isoforms of ShcA were phosphorylated in response to NGF in DRG and PC12 cells. NGF phosphorylated only one whereas epidermal growth factor phosphorylated both isoforms of ShcC in DRG cultures. Activation of the downstream mitogen-activated protein (MAP) kinase, p42Erk2 was significantly greater than p44Erk1 in DRG whereas both isoforms were activated in PC12 cells. Blocking the MAP kinase cascade using a MEK1/2 inhibitor, PD98059, abrogated NGF dependent capsaicin sensitivity, a nociceptive property specific to sensory neurons.     

PC12h-R cell,a subclone of PC12 cells,shows EGF-induced neuronal differentiation and sustained signaling

Unlike nerve growth factor (NGF), epidermal growth factor (EGF) does not induce neuronal differentiation but promotes proliferation of the rat pheochromocytoma PC12 cells. We found that PC12h-R, a subclone of PC12 cells, differentiated into neuron-like cells in response to EGF as well as to NGF. PC12h-R cells treated with EGF extended neurites, attenuated cell proliferation, and increased the levels of tyrosine hydroxylase protein synthesis and of acetylcholinesterase activity as those treated with NGF. The EGF-induced differentiation of PC12h-R cells was not mediated by the indirect activation of p140^trkA by EGF. In addition, EGF induced the sustained tyrosine phosphorylation of the EGF receptor, mitogen-activated protein (MAP) kinases, and 46 and 52 kDa proteins, and the prolonged activation of MAP kinases in PC12h-R cells compared with the parent PC12h, which does not show EGF-induced differentiation. The response of PC12h-R cells to EGF was not simply due to an increase in the level of EGF receptor protein. These results indicated that the duration of EGF-induced signaling might determine the cellular response of PC12 cells between cell proliferation and neuronal differentiation. © 1996 Wiley-Liss, Inc.     

Identification of amino acid residues of nerve growth factor important for neurite outgrowth in human dorsal root ganglion neurons

Nerve growth factor (NGF) is an essential neurotrophic factor for the development and maintenance of the central and the peripheral nervous system. NGF deficiency in the basal forebrain precedes degeneration of basal forebrain cholinergic neurons in Alzheimer's disease, contributing to memory decline. NGF mediates neurotrophic support via its high‐affinity receptor, the tropomyosin‐related kinase A (TrkA) receptor, and mediates mitogenic and differentiation signals via the extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2). However, the molecular mechanisms underlying the different NGF/TrkA/ERK signalling pathways are far from clear. In this study, we have investigated the role of human NGF and three NGF mutants, R100E, W99A and K95A/Q96A, their ability to activate TrkA or ERK1/2, and their ability to induce proliferation or differentiation in human foetal dorsal root ganglion (DRG) neurons or in PC12 cells. We show that the R100E mutant was significantly more potent than NGF itself to induce proliferation and differentiation, and significantly more potent in activation of ERK1/2 in DRG neurons. The W99A and K95A/Q96A mutants, on the other hand, were less effective than the wild‐type protein. An unexpected finding was the high efficacy of the K95A/Q96A mutant to activate TrkA and to induce differentiation of DRG neurons at elevated concentrations. These data demonstrate an NGF mutant with improved neurotrophic properties in primary human neuronal cells. The R100E mutant represents an interesting candidate for further drug development in Alzheimer's disease and other neurodegenerative disorders.     

Novel functional interactions between Trk kinase and p75 neurotrophin receptor in neuroblastoma cells

To understand the functional interactions between the TrkA and p75 nerve growth factor (NGF) receptors, we stably transfected LAN5 neuroblastoma cells with an expression vector for ET-R, a chimeric receptor with the extracellular domain of the epidermal growth factor receptor (EGFR), and the TrkA transmembrane and intracellular domains. EGF activated the ET-R kinase and induced partial differentiation. NGF, which can bind to endogenous p75, did not induce differentiation but enhanced the EGF-induced response, leading to differentiation of almost all cells. A mutated NGF, 3T-NGF, that binds to TrkA but not to p75 did not synergize with EGF. Enhancement of EGF-induced differentiation required at least nanomolar concentrations of NGF, consistent with the low-affinity p75 binding site. EGF may induce a limited number of neuronal cells because it also enhanced apoptosis. Both NGF and a caspase inhibitor reduced apoptosis and, thereby, enhanced differentiation. NGF seems to enhance survival through the phosphatidylinositol-3 kinase (PI3K) pathway. Consistent with this hypothesis, Akt, a downstream effector of the PI3K pathway, was hyperphosphorylated in the presence of EGF+NGF. These results demonstrate that TrkA kinase initiates differentiation, and p75 enhances differentiation by rescuing differentiating cells from apoptosis via the PI3K pathway. Even though both EGF and NGF are required for differentiation of LAN5/ET-R cells, only NGF is required for survival of the differentiated cells. In the absence of NGF, the cells die by an apoptotic mechanism, involving caspase-3. An anti-p75 antibody blocked the survival effect of NGF. Brain-derived neurotrophic factor also enhanced cell survival, indicating that in differentiated cells, NGF acts through the p75 receptor to prevent apoptosis.