Programmed cell death in the developing somites is promoted by nerve growth factor via its p75(NTR) receptor

Neurotrophins control neuron number during development by promoting the generation and survival of neurons and by regulating programmed neuronal death. In the latter case, the cell death induced by nerve growth factor (NGF) in the developing chick retina is mediated by p75(NTR), the common neurotrophin receptor (J. M. Frade, A. Rodriguez-Tebar, and Y.-A. Barde, 1996, Nature 383, 166-168). Here we show that NGF also induces the programmed death of paraxial mesoderm cells in the developing somites. Both NGF and p75(NTR) are expressed in the somites of chick embryos at the time and the place of programmed cell death. Moreover, neutralizing the activity of endogenous NGF with a specific blocking antibody, or antagonizing NGF binding to p75(NTR) by the application of human NT-4/5, reduces the levels of apoptotic cell death in both the sclerotome and the dermamyotome by about 50 and 70%, respectively. Previous data have shown that Sonic hedgehog is necessary for the survival of differentiated somite cells. Consistent with this, Sonic hedgehog induces a decrease of NGF mRNA in somite explant cultures, thus showing the antagonistic effect of NGF and Sonic hedgehog with respect to somite cell survival. The regulation of programmed cell death by NGF/p75(NTR) in a mesoderm-derived tissue demonstrates the capacity of neurotrophins and their receptors to influence critical developmental processes both within and outside of the nervous system.     

Individual and combined effects of TrkA and p75NTR nerve growth factor receptors. A role for the high affinity receptor site

A long-standing question in neurotrophin signal transduction is whether heteromeric TrkA-p75NTR complexes possess signaling capabilities that are significantly different from homo-oligomeric TrkA or p75NTR alone. To address this issue, various combinations of transfected PC12 cells expressing a platelet-derived growth factor receptor-TrkA chimera and the p75NTR-selective nerve growth factor mutant (Delta9/13 NGF) were utilized to selectively stimulate TrkA or p75NTR signaling, respectively. The contribution of individual and combined receptor effects was analyzed in terms of downstream signaling and certain end points. The results suggest two unique functions for the high affinity heteromeric NGF receptor site: (a) integration of both the MAPK and Akt pathways in the production of NGF-induced neurite outgrowth, and (b) rapid and sustained activation of the Akt pathway, with consequent long term cellular survival. Whereas activation of TrkA signaling is sufficient for eliciting neurite outgrowth in PC12 cells, signaling through p75NTR plays a modulatory role, especially in the increased formation of fine, synaptic "bouton-like" structures, in which both TrkA and p75NTR appear to co-localize. In addition, a new interaction in the TrkA/p75NTR heteromeric receptor signal transduction network was revealed, namely that NGF-induced activation of the MAPK pathway appears to inhibit the parallel NGF-induced Akt pathway.     

Evidence for a critical role of the tumor necrosis factor alpha convertase (TACE) in ectodomain shedding of the p75 neurotrophin receptor (p75NTR)

Protein ectodomain shedding, the proteolytic release of the extracellullar domain of membrane-tethered proteins, can dramatically affect the function of cell surface receptors, growth factors, cytokines, and other proteins. In this study, we evaluated the activities involved in ectodomain shedding of p75NTR, a neurotrophin receptor with critical roles in neuronal differentiation and survival. p75NTR is shed in a variety of cell types, including dorsal root ganglia cells and PC12 cells. In Chinese hamster ovary cells, inhibitors of the MEK/ERK and p38 MAP kinase pathways uncovered distinct signaling pathways required for the constitutive and stimulated shedding of p75NTR. Stimulated p75NTR shedding is abrogated in M2 mutant Chinese hamster ovary cells that lack functional tumor necrosis factor-alpha converting enzyme (TACE, also referred to as ADAM17) and in cells isolated from adam17-/- mice, but not in cells from adam9/12/15-/- or adam10-/- mice. Stimulated p75(NTR) shedding is strongly reduced by deletion of 15 amino acid residues in its extracellular membrane-proximal stalk domain. However, similar to other shed proteins, point mutations and overlapping shorter deletions within this region have little or no effect on shedding. Because ectodomain shedding of p75NTR releases a soluble ectodomain and could also be a prerequisite for its regulated intramembrane proteolysis, these findings may have important implications for the functional regulation of p75NTR.     

Evidence for the participation of nerve growth factor and its low-affinity receptor (p75NTR) in the regulation of the myogenic program

We have studied expression and function of neurotrophins and their receptors during myogenic differentiation of C2C12 cells, a clonal cell line derived from mouse muscle that is capable of in vitro differentiation. The genes coding for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their common low-affinity receptor p75^{neurotrophin receptor} (p75^NTR) were shown to be expressed in C2C12 myoblasts and downregulated during myogenic differentiation and fusion into myotubes. Cocultures with dorsal root ganglia from day 8 chick embryos revealed neurite-promoting activities of C2C12 cells that ceased with myogenic differentiation. These data suggest a temporal and developmental window for the effect of myogenic cell-derived neurotrophins on neuronal as well as on myogenic cell populations. NGF was shown to increase DNA synthesis and cell growth of C2C12 myoblasts and to enhance myogenic differentiation in this cell line. We present evidence that NGF-mediated processes take place at stages preceding myogenic differentiation. Enhanced muscle differentiation was also seen in p75^NTR-overexpressing C2C12 myoblasts which maintained high levels of receptors but ceased to produce NGF during differentiation. In contrast, when exogenous NGF was present at the onset of myogenic differentiation of receptor-overexpressing cells, muscle cell development was strongly repressed. This indicates that downregulation of p75^NTR is necessary for guiding myogenic cells towards terminal differentiation. Since none of the trk high-affinity neurotrophin receptors could be demonstrated in C2C12 cells, we conclude that NGF mediates its nonneurotrophic effect via its low-affinity receptor in an autocrine fashion. J. Cell. Physiol. 176:10–21, 1998. © 1998 Wiley-Liss, Inc.     

p75NTR signal transduction suppressed by BFAR and p75NTR interactions

p75NTR is a low-affinity nerve growth factor receptor, which promotes cell proliferation as a positive modulator of high-affinity receptor TrkA, as well as binds with cell ligands to induce apoptosis and mediate death signals. To analyze the regulatory mechanisms of p75NTR, the present study utilized a new membrane yeast two-hybrid system to screen a human fetal brain cDNA library. Results identified BFAR, a novel protein that interacts with p75NTR. Interaction specificity was verified by membrane yeast two-hybrid co-transformation assays, in vitro GST pull-down assays, and in vitro co-immunoprecipitation assays. The fluorescent subcellular localization assay revealed that the two proteins co-localized within the cytoplasm. BFAR overexpression in PC-12 and HEK293T cells inhibited the NFκB and JNK signaling pathway, as determined with the luciferase test. Co-transfected p75NTR and BFAR in HEK293T or PC-12 cells, respectively, increased the percentage of cells in the G2/M phase, decreased the number of S-phase cells, and did not change the number of G0/G1-phase cells.     

TrkA receptor activation by nerve growth factor induces shedding of the p75 neurotrophin receptor followed by endosomal gamma-secretase-mediated release of the p75 intracellular domain

Neurotrophins are trophic factors that regulate important neuronal functions. They bind two unrelated receptors, the Trk family of receptor-tyrosine kinases and the p75 neurotrophin receptor (p75). p75 was recently identified as a new substrate for gamma-secretase-mediated intramembrane proteolysis, generating a p75-derived intracellular domain (p75-ICD) with signaling capabilities. Using PC12 cells as a model, we studied how neurotrophins activate p75 processing and where these events occur in the cell. We demonstrate that activation of the TrkA receptor upon binding of nerve growth factor (NGF) regulates the metalloprotease-mediated shedding of p75 leaving a membrane-bound p75 C-terminal fragment (p75-CTF). Using subcellular fractionation to isolate a highly purified endosomal fraction, we demonstrate that p75-CTF ends up in endosomes where gamma-secretase-mediated p75-CTF cleavage occurs, resulting in the release of a p75-ICD. Moreover, we show similar structural requirements for gamma-secretase processing of p75 and amyloid precursor protein-derived CTFs. Thus, NGF-induced endocytosis regulates both signaling and proteolytic processing of p75.     

Glucose homeostasis and p75NTR

Comment on: Baeza-Raja B, et al. Proc Natl Acad Sci USA 2012; 109:5838-43.     

Characterization of the human melanoma nerve growth factor receptor

Monoclonal antibodies to the human nerve growth factor receptor have been used to biochemically characterize the receptor in the human melanoma cell line A875. Labeling of A875 cell proteins by culture with [35S]cysteine or labeling of cell surface proteins with 125I followed by immunoprecipitation with anti-nerve growth factor receptor antibody reveals a receptor protein with an apparent Mr of 70,000-75,000 and an isoelectric point of 4.9-5.2. Incorporation of [3H]glucosamine into this species indicates it is a glycoprotein. The receptor becomes phosphorylated on serine residues in intact cells and in isolated membranes incubated with [gamma-32P]ATP. The receptor appears to exist, at least partially, in the form of a disulfide-linked oligomer (probably a dimer) of Mr = 75,000 subunits. Kinetic [35S]cysteine labeling studies reveal an Mr = 59,000 core protein which is glycosylated via N-linked and probably also O-linked sugar moieties to produce the mature (Mr = 70,000-75,000) receptor.     

Glucose regulates expression of the nerve growth factor (NGF) receptors TrkA and p75NTR in rat islets and INS-1E beta-cells

BACKGROUND: The function and survival of pancreatic beta-cells strongly depend on glucose concentration and on autocrine secretion of peptide growth factors. NGF and its specific receptors TrkA and p75NTR play a pivotal role in islet survival and glucose-dependent insulin secretion. We therefore investigated whether or not glucose concentration influences expression of TrkA and p75NTR in rat islets and in INS-1E beta-cells at the mRNA and protein level (INS-1E). METHODS: Gene expression of the NGF receptors TrkA and p75NTR but also of the metabolic gene liver-type pyruvate kinase (L-PK) and the neurotrophin receptors TrkB and TrkC was studied by semi-quantitative PCR and by real-time PCR in islets and INS-1E beta-cells. RESULTS: In rat islets, high glucose exposure (25 mmol/l) increased gene expression of TrkA, p75NTR and L-PK. Expression of TrkA, p75NTR and L-PK reflected insulin secretion at the respective glucose concentration. In rat INS-1E insulinoma cells, expression of L-PK and p75NTR was suppressed by low glucose as in the islets, while expression of TrkA was strongly increased by low glucose levels and thus was regulated differently than in islets. Expression of TrkB and TrkC was not regulated by glucose concentration at all. TrkA protein was regulated in the same fashion as its mRNA expression, while p75NTR protein was not significantly regulated within 24 h. CONCLUSION: Glucose interacts with gene expression of TrkA and p75NTR that are strongly involved in beta-cell growth and glucose-dependent insulin secretion. The fact that TrkA expression is regulated the opposite way in islets and in INS-1E beta-cells might reflect their specific grade of differentiation and tendency to proliferate.     

Shedding of the p75NTR neurotrophin receptor is modulated by lipid rafts

Protein ectodomain shedding is the proteolytic release of the extracellular domain of membrane-bound proteins. Neurotrophin receptor p75(NTR) is known to be affected by shedding. The present work provides evidence, in rat brain synaptosomes, that p75(NTR) is present in detergent-resistant membranes (DRM), also known as lipid rafts, only in its full-length form. Disrupting the integrity of lipid rafts causes solubilization of p75(NTR) after detergent treatment and enhancement of the shedding. Analyses of the enzymes described as being responsible for p75(NTR) shedding, i.e. tumor necrosis factor alpha convertase (TACE) and presenilin-1 (PS1), revealed that TACE is absent in DRM, while variable proportions of the C-terminal and N-terminal fragments of PS1 are found. In summary, our results point to a role of lipid rafts in the modulation of the shedding of the p75(NTR) receptor.     

The many faces of p75NTR

The neurotrophins utilize a complex receptor system consisting of Trk receptor tyrosine kinases and the structurally unrelated tumor necrosis factor receptor family member, p75(NTR), to confer diverse and sometimes opposing biological actions. The recent identification of selective ligands for p75(NTR), novel isoforms of this receptor, as well as new signaling partners, suggest that the numerous biological actions of the neurotrophins via p75(NTR) may reflect selectivity of ligand-receptor interactions and intracellular adaptor protein recruitment.     

Proteome changes induced by overexpression of the p75 neurotrophin receptor (p75NTR) in breast cancer cells

In breast cancer cells, the neurotrophin receptor p75(NTR) acts as a prosurvival factor able to stimulate resistance to apoptosis, but its mechanism of action remains incompletely defined. In this study, we investigated the global proteome modification induced by p75(NTR) overexpression in breast cancer cells treated by the pro-apoptotic agent tumor necrosis factor (TNF)-related-apoptosis-inducing-ligand (TRAIL). p75(NTR) was stably overexpressed in the MCF-7 breast cancer cells and the impact of a treatment by TRAIL was investigated in wild type vs. p75(NTR) overexpressing cells. Proteins were separated in two-dimensional electrophoresis, and regulated spots were detected by computer assisted analysis before identification by MALDI-TOF/TOF mass spectrometry. In the absence of TRAIL treatment, p75(NTR) did not induce any change in the proteome of breast cancer cells. In contrast, after treatment with TRAIL, fragments of cytokeratin-8, -18 and -19, as well as full length cytokeratin-18, were up-regulated by p75(NTR) overexpression. Of note, spectrin alpha-chain and the ribosomal protein RPLP0 were induced by TRAIL, independently of p75(NTR) level. Interestingly, the well known stress-induced protein HSP-27 was less abundant when p75(NTR) was overexpressed, indicating that p75(NTR) overexpression reduced TRAIL induced cell stress. These data indicate that overexpression of p75(NTR) induces proteome modifications in breast cancer cells and provide information on how this receptor contributes in tumor cell resistance to apoptosis.     

NADE, a p75NTR-associated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR

The low affinity neurotrophin receptor p75NTR can mediate cell survival as well as cell death of neural cells by NGF and other neurotrophins. To elucidate p75NTR-mediated signal transduction, we screened p75NTR-associated proteins by a yeast two-hybrid system. We identified one positive clone and named NADE (p75NTR-associated cell death executor). Mouse NADE has marked homology to the human HGR74 protein. NADE specifically binds to the cell-death domain of p75NTR. Co-expression of NADE and p75NTR induced caspase-2 and caspase-3 activities and the fragmentation of nuclear DNA in 293T cells. However, in the absence of p75NTR, NADE failed to induce apoptosis, suggesting that NADE expression is necessary but insufficient for p75NTR-mediated apoptosis. Furthermore, p75NTR/NADE-induced cell death was dependent on NGF but not BDNF, NT-3, or NT-4/5, and the recruitment of NADE to p75NTR (intracellular domain) was dose-dependent. We obtained similar results from PC12 cells, nnr5 cells, and oligodendrocytes. Taken together, NADE is the first signaling adaptor molecule identified in the involvement of p75NTR-mediated apoptosis induced by NGF, and it may play an important role in the pathogenesis of neurogenetic diseases.     

Disruption of cysteine-rich repeats of the p75 nerve growth factor receptor leads to loss of ligand binding.

Nerve growth factor (NGF) binds to a low affinity cell surface receptor (p75NGFR) which contains four extracellular repeats, rich in cysteine residues and negatively charged. We have made mutations in the receptor cDNA by inserting linkers in specific domains of the receptor. Nearly all the mutations caused a change in the predicted charge, and resulted in either an insertion or deletion in the primary sequence. Stably transfected fibroblasts were assayed for NGF binding by affinity cross-linking with 125I-NGF. Appropriate expression of the mutated receptors was monitored by rosetting with monoclonal antibodies and by metabolic labeling followed by immunoprecipitation. Although the mutant receptors were recognized by monoclonal antibodies, insertions and deletions in the third and fourth cysteine-rich regions of the receptor had a detrimental effect upon NGF binding. Insertions made outside the cysteine-rich region or in the cytoplasmic domain did not inhibit the ability of 125I-NGF to bind to the receptor, as assessed by affinity cross-linking. A chimeric human-rat NGF receptor transfected into fibroblasts indicates that NGF binding and monoclonal antibody recognition sites are separated but contained within the four cysteine repeats.