The p75(NTR) tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

The p75 neurotrophin receptor (p75(NTR)) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75(NTR) retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted (DeltaDD) dominant-negative antagonist of p75(NTR) showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75(NTR)-dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75(NTR) expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75(NTR) rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75(NTR) was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75(NTR)-dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75(NTR) expressing prostate cancer cells.     

Effect of p75NTR on the regulation of naturally occurring cell death and retinal ganglion cell number in the mouse eye

Neurotrophins induce neural cell survival and differentiation during retinal development and regeneration through the high-affinity tyrosine kinase (Trk) receptors. On the other hand, nerve growth factor (NGF) binding to the low-affinity neurotrophin receptor p75 (p75(NTR)) might induce programmed cell death (PCD) in the early phase of retinal development. In the present study, we examined the retinal cell types that experience p75(NTR)-induced PCD and identify them to be postmitotic retinal ganglion cells (RGCs). However, retinal morphology, RGC number, and BrdU-positive cell number in p75(NTR) knockout (KO) mouse were normal after embryonic day 15 (E15). In chick retina, migratory RGCs express p75(NTR), whereas layered RGCs express the high-affinity NGF receptor TrkA, which may switch the pro-apoptotic signaling of p75(NTR) into a neurotrophic one. In contrast to the chick model, migratory RGCs express TrkA, while stratified RGCs express p75(NTR) in mouse retina. However, RGC number in TrkA KO mouse was also normal at birth. We next examined the expression of transforming growth factor beta (TGFbeta) receptor, which modulates chick RGC number in combination with p75(NTR), but was absent in mouse RGCs. p75(NTR) and TrkA seem to be involved in the regulation of mouse RGC number in the early phase of retinal development, but the number may be later adjusted by other molecules. These results suggest the different mechanism of RGC number control between mouse and chick retina.     

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.     

The interaction of neurotrophins with the p75NTR common neurotrophin receptor: a comprehensive molecular modeling study

Neurotrophins are a family of proteins with pleiotropic effects mediated by two distinct receptor types, namely the Trk family, and the common neurotrophin receptor p75NTR. Binding of four mammalian neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), to p75NTR is studied by molecular modeling based on X-ray structures of the neurotrophins and the extracellular domain of p55TNFR, a homologue of p75NTR. The model of neurotrophin/receptor interactions suggests that the receptor binding domains of neurotrophins (loops I and IV) are geometrically and electrostatically complementary to a putative binding site of p75NTR, formed by the second and part of the third cysteine-rich domains. Geometric match of neurotrophin/receptor binding domains in the complexes, as characterized by shape complementarity statistic Sc, is comparable to known protein/protein complexes. All charged residues within the loops I and IV of the neurotrophins, previously determined as being critical for p75NTR binding, directly participate in receptor binding in the framework of the model. Principal residues of the binding site of p75NTR include Asp47, Lys56, Asp75, Asp76, Asp88, and Glu89. The additional involvement of Arg80 and Glu53 is specific for NGF and BDNF, respectively, and Glu73 participates in binding with NT-3 and NT-4/5. Neurotrophins are likely to induce similar, but not identical, conformational changes within the p75NTR binding site.     

CD 271 (P75 neurotrophin receptor)

P75NTR (or CD271) is a member of the Tumor Necrosis Factor receptor (TNFR) super family of transmembrane proteins that share significant homology in their extracellular domains. Subsets of TNF receptors, including CD271, have a cytoplasmic death domain, although CD271 has unique intracellular structure and downstream signaling partners. CD271 is also differentiated from other members of the TNFR receptor family in that it binds pro and mature neurotrophins and affects the growth, differentiation and death of the nervous system. The ligands for CD271 are neurotrophins, which are Nerve Growth Factor (NGF), Brain-Derived Growth factor (BDNF), Neurotrophin 3 (NT3) and Neurotrophin 4/5 (NT4/5). Recent studies have provided evidence that CD271 also serves as a receptor for the pro-forms of these neurotrophins.     

p75NTR: A study in contrasts

The p75 neurotrophin receptor (p75NTR) and trkA, trkB and trkC mediate the physiological effects of the neurotrophins. The trk receptors are responsible for the stereotypical survival and growth properties of the neurotrophins but defining the physiological function of the p75NTR has proven difficult. The p75NTR binds each of the neurotrophins with low nanomolar affinity whereas the three trk receptors show strong binding preferences for individual neurotrophins; in some cell types, p75NTR is the only neurotrophin receptor whereas in others it is co-expressed with the trks. The analysis of p75NTR function has been complicated by the fact that the predominant physiological role of p75NTR changes dramatically depending on cell context. Available data suggests that in cells where p75NTR is co-expressed with trk receptors, p75NTR functionally collaborates with the trks to either enhance responses to preferred trk ligands, to reduce neurotrophin-mediated trk receptor activation resulting from non-preferred ligands or to facilitate apoptosis resulting from neurotrophin withdrawal. In cells lacking trk expression, p75NTR can act autonomously to activate ligand-dependent signaling cascades that may in some circumstances result in apoptosis but probably not through pathways utilized by its apoptotic brethren in the TNF receptor superfamily. Potential mechanisms for each of these functions of p75NTR are considered and the physiological implications of this unique signaling system are discussed.     

Haloperidol and atypical antipsychotics share a same action of decreasing P75(NTR) mRNA levels in PC12 cells

P75(NTR) is a common neurotrophin receptor which binds all neurotrophins with similar affinities and has been shown to be capable of mediating programmed cell death. In this study, we investigated effects of the antipsychotic drugs (APDs) haloperidol, clozapine, quetiapine, and risperidone on p75(NTR) mRNA levels in PC12 cells. Haloperidol is a prototype of typical APDs, and the other three drugs are atypical APDs, which are effective in reducing negative symptoms and cognitive deficits of schizophrenia, cause less side effects, and are more tolerable compared to haloperidol. PC12 cells were cultured with various concentrations of haloperidol, clozapine, quetiapine, or risperidone, in their media. After culture for 48h, the cell viabilities and p75(NTR) mRNA levels were measured. It was shown that both haloperidol and the atypical APDs used in this study deceased p75(NTR) mRNA levels in PC12 cells in a dose dependent manner, while not affecting cell viabilities. In further experiments, doses that produced significant/greatest effects were chosen and provided in the culture media for various periods. Decreases in p75(NTR) mRNA levels were observed in cultures treated for 12h with quetiapine, 24h with clozapine or risperidone, or for 48h with haloperidol. These results suggest that both haloperidol and atypical APDs have the same action of decreasing p75(NTR) mRNA levels in PC12 cells. Although the underlying molecular mechanism of this action remains to be elucidated, this finding is particularly relevant given the neurodevelopmental deficits associated with schizophrenia and important roles of p75(NTR) in mediating cell death.     

Proteolytic processing of the p75 neurotrophin receptor: A prerequisite for signalling?

The common neurotrophin receptor (p75(NTR) ) regulates various functions in the developing and adult nervous system. Cell survival, cell death, axonal and growth cone retraction, and regulation of the cell cycle can be regulated by p75(NTR) -mediated signals following activation by either mature or pro-neurotrophins and in combination with various co-receptors, including Trk receptors and sortilin. Here, we review the known functions of p75(NTR) by cell type, receptor-ligand combination, and whether regulated intra-membrane proteolysis of p75(NTR) is required for signalling. We highlight that the generation of the intracellular domain fragment of p75(NTR) is associated with many of the receptor functions, regardless of its ligand and co-receptor interactions.     

The genome sequence of the protostome Daphnia pulex encodes respective orthologues of a neurotrophin, a Trk and a p75NTR: Evolution of neurotrophin signaling components and related proteins in the bilateria

Background

Neurotrophins and their Trk and p75NTR receptors play an important role in the nervous system. To date, neurotrophins, Trk and p75NTR have only been found concomitantly in deuterostomes. In protostomes, homologues to either neurotrophin, Trk or p75NTR are reported but their phylogenetic relationship to deuterostome neurotrophin signaling components is unclear. Drosophila has neurotrophin homologues called Spätzles (Spz), some of which were recently renamed neurotrophins, but direct proof that these are deuterostome neurotrophin orthologues is lacking. Trks belong to the receptor tyrosine kinase (RTK) family and among RTKs, Trks and RORs are closest related. Flies lack Trks but have ROR and ROR-related proteins called NRKs playing a neurotrophic role. Mollusks have so far the most similar proteins to Trks (Lymnaea Trk and Aplysia Trkl) but the exact phylogenetic relationship of mollusk Trks to each other and to vertebrate Trks is unknown. p75NTR belongs to the tumor necrosis factor receptor (TNFR) superfamily. The divergence of the TNFR families in vertebrates has been suggested to parallel the emergence of the adaptive immune system. Only one TNFR representative, the Drosophila Wengen, has been found in protostomes. To clarify the evolution of neurotrophin signaling components in bilateria, this work analyzes the genome of the crustacean Daphnia pulex as well as new genetic data from protostomes.

Results

The Daphnia genome encodes a neurotrophin, p75NTR and Trk orthologue together with Trkl, ROR, and NRK-RTKs. Drosophila Spz1, 2, 3, 5, 6 orthologues as well as two new groups of Spz proteins (Spz7 and 8) are also found in the Daphnia genome. Searching genbank and the genomes of Capitella, Helobdella and Lottia reveals neurotrophin signaling components in other protostomes.

Conclusion

It appears that a neurotrophin, Trk and p75NTR existed at the protostome/deuterostome split. In protostomes, a "neurotrophin superfamily" includes Spzs and neurotrophins which respectively form two paralogous families. Trks and Trkl proteins also form closely related paralogous families within the protostomian RTKs, whereby Trkls are absent in deuterostomes. The finding of p75NTR in several protostomes suggests that death domain TNFR superfamily proteins appeared early in evolution.     

Control of the cell cycle by neurotrophins: lessons from the p75 neurotrophin receptor

Although traditionally little attention has been paid to the interplay between neurotrophins and the cell cycle, a number of recent findings suggest an important role for these growth factors in the regulation of this aspect of the cellular physiology. In this article, we review the evidence from a number of studies that neurotrophins can influence cell cycle progression or mitotic cycle arrest both in the nervous system as well as in other cell types. The contrary response of different cells to neurotrophins in terms of cell cycle regulation derives in part from the fact that these factors use two different receptor types to transmit their signals: members of the Trk family and the p75 neurotrophin receptor (p75NTR). With this in mind, we outline the current state of our knowledge regarding the molecular basis underlying the control of cell cycle progression by neurotrophins. We focus our interest on the receptors that transduce these signals and, in particular, the striking finding that p75NTR interacts with proteins that can promote mitotic cycle arrest. Finally, we discuss the mechanisms of cell death mediated by p75NTR in the context of cell cycle regulation.     

Neurotrophin dependence mediated by p75NTR: contrast between rescue by BDNF and NGF

During development, neurons pass through a critical phase in which survival is dependent on neurotrophin support. In order to dissect the potential role of p75NTR, the common neurotrophin receptor, in neurotrophin dependence, we expressed wild-type and mutant p75NTR in cells that do not express endogenous p75NTR or Trk family members (NIH3T3). Expression of wild-type p75NTR created a state of neurotrophin dependence: cells could be rescued by nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3), but not by a mutant NGF that binds well to Trk A but poorly to p75NTR. Similarly, expression of p75NTR in human prostate cancer cells in culture rendered a metastatic tumor cell line (PC-3) sensitive to the availability of neurotrophins for survival. Moreover, expression of mutant p75NTR's in another neurotrophin-insensitive cell line (HEK293T) showed that a domain within the intracellular domain governs alternate responses to neurotrophins: the carboxy terminus of the intracellular domain of p75NTR including the sixth alpha helix domain is necessary for rescue by BDNF, but not NGF. These results, when considered with previous studies of the timing of p75NTR expression, support the hypothesis that p75NTR is a mediator of neurotrophin dependence during the critical phase of developmental cell death and during the progression of carcinogenesis in prostate cancer.     

Single chain TNF derivatives with individually mutated receptor binding sites reveal differential stoichiometry of ligand receptor complex formation for TNFR1 and TNFR2

Most members of the tumor necrosis factor ligand family form noncovalently linked homotrimers, capable to bind up to three molecules of the respective membrane receptors. For several receptors a membrane distal homophilic interaction domain has been identified, called pre-ligand binding assembly domain. Accordingly, affinity values determined by typical equilibrium binding studies are likely to be influenced by avidity effects. Using our recently introduced covalently stabilized TNF (single chain TNF, scTNF), we have here investigated receptor–ligand binding stoichiometry in our well characterized system of TNFR–Fas chimeras. We produced scTNF derivatives with functionally deleted individual receptor binding sites, resulting in TNF mutants capable to only bind to one or two receptor molecules, rather than three. Equilibrium binding affinity studies on ice with these molecules revealed no significant changes after a single receptor binding site had been functionally deleted. In contrast, functional abrogation of two receptor binding sites showed a strong decrease in both, affinity and bioactivity on TNFR2–Fas. In contrast, TNFR1–Fas ligand binding and receptor activation was only affected after functional deletion of all three receptor binding sites. Our data demonstrate pivotal differences in ligand/receptor interactions between TNFR1–Fas and TNFR2–Fas, arguing for avidity effects important for TNF binding and downstream signaling of TNFR2, but to a lesser extent of TNFR1. These results are supported by data revealed from chemical crosslinking experiments suggesting the existence of preformed TNFR–Fas homodimers.     

p75 neurotrophin receptor-mediated neuronal death is promoted by Bcl-2 and prevented by Bcl-xL.

The p75 neurotrophin receptor (p75NTR) has been shown to mediate neuronal death through an unknown pathway. We microinjected p75NTR expression plasmids into sensory neurons in the presence of growth factors and assessed the effect of the expressed proteins on cell survival. We show that, unlike other members of the TNFR family, p75NTR signals death through a unique caspase-dependent death pathway that does not involve the "death domain" and is differentially regulated by Bcl-2 family members: the anti-apoptotic molecule Bcl-2 both promoted, and was required for, p75NTR killing, whereas killing was inhibited by its homologue Bcl-xL. These results demonstrate that Bcl-2, through distinct molecular mechanisms, either promotes or inhibits neuronal death depending on the nature of the death stimulus.     

NRAGE,a p75 neurotrophin receptor-interacting protein,induces caspase activation and cell death through a JNK-dependent mitochondrial pathway

The p75 neurotrophin receptor (p75NTR) mediates signaling events leading to activation of the JNK pathway and cell death in a variety of cell types. We recently identified NRAGE, a protein that directly interacts with the p75NTR cytosolic region and facilitates p75NTR-mediated cell death. For the present study, we developed an inducible recombinant NRAGE adenovirus to dissect the mechanism of NRAGE-mediated apoptosis. Induced NRAGE expression resulted in robust activation of the JNK pathway that was not inhibited by the pharmacological mixed lineage kinase (MLK) inhibitor CEP1347. NRAGE induced cytosolic accumulation of cytochrome c, activation of Caspases-3, -9 and -7, and caspase-dependent cell death. Blocking JNK and c-Jun action by overexpression of the JNK-binding domain of JIP1 or dominant-negative c-Jun ablated NRAGE-mediated caspase activation and NRAGE-induced cell death. These findings identify NRAGE as a p75NTR interactor capable of inducing caspase activation and cell death through a JNK-dependent mitochondrial apoptotic pathway.     

Sortilin participates in light-dependent photoreceptor degeneration in vivo

Both proNGF and the neurotrophin receptor p75 (p75(NTR)) are known to regulate photoreceptor cell death caused by exposure of albino mice to intense illumination. ProNGF-induced apoptosis requires the participation of sortilin as a necessary p75(NTR) co-receptor, suggesting that sortilin may participate in the photoreceptor degeneration triggered by intense lighting. We report here that light-exposed albino mice showed sortilin, p75(NTR), and proNGF expression in the outer nuclear layer, the retinal layer where photoreceptor cell bodies are located. In addition, cone progenitor-derived 661W cells subjected to intense illumination expressed sortilin and p75(NTR) and released proNGF into the culture medium. Pharmacological blockade of sortilin with either neurotensin or the "pro" domain of proNGF (pro-peptide) favored the survival of 661W cells subjected to intense light. In vivo, the pro-peptide attenuated retinal cell death in light-exposed albino mice. We propose that an auto/paracrine proapoptotic mechanism based on the interaction of proNGF with the receptor complex p75(NTR)/sortilin participates in intense light-dependent photoreceptor cell death. We therefore propose sortilin as a putative target for intervention in hereditary retinal dystrophies.     

TNF receptor 2-deficient CD8 T cells are resistant to Fas/Fas ligand-induced cell death

Apoptotic cell death plays a fundamental role in the maintenance of tissue homeostasis in complex biological systems. It is also a major mechanism for keeping immune reactions in check. Members of the TNF family of receptors and cytokines are implicated in the regulation of apoptotic signals that shape the immune system. In this study, we have examined the role of three members of the TNFR family, Fas (CD95), TNFR1 (p55), and TNFR2 (p75), in inducing cell death in Con A-activated CD4 and CD8 T cells. It was found that Con A-activated p55(-/-) CD4 or CD8 T cells were highly resistant to TNF-induced cell death. By contrast, although activated p75(-/-) CD4 or CD8 T cells were killed by TNF, they were more resistant to TNF-induced killing when compared with p75(+/+) cells, particularly at higher concentrations of TNF. We also determined whether activated p55(-/-) and p75(-/-) T cells differ in their sensitivity to cell death induced by TCR cross-linking. We found that activated p55(-/-) CD4 or CD8 T cells were equally susceptible to TCR-induced cell death. More interestingly, the loss of the p75 receptor conferred resistance to TCR-induced death in activated CD8, but not CD4 T cells. This resistance to TCR-induced death in activated p75(-/-) CD8 T cells correlated with the resistance of these cells to Fas/Fas ligand-induced cell death.     

Interactions between beta-neuregulin and neurotrophins in motor neuron apoptosis

Neuregulins play a major role in the formation and stabilization of neuromuscular junctions, and are produced by both motor neurons and muscle. Although the effects and mechanism of neuregulins on skeletal muscle (e.g. regulation of acetylcholine receptor expression) have been studied extensively, the effects of neuregulins on motor neurons remain unknown. We report that neuregulin-1beta (NRGbeta1) inhibited apoptosis of rat motor neurons for up to 7 days in culture by a phosphatidylinositol 3 kinase-dependent pathway and synergistically enhanced motor neuron survival promoted by glial-derived neurotrophic factor (GDNF). However, binding of neurotrophins, including brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), to the p75 neurotrophin receptor (p75NTR) abolished the neuregulin anti-apoptotic effect on motor neurons. Inhibitors of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase prevented motor neuron death caused by co-incubation of NRGbeta1 and BDNF or NGF, as well as by trophic factor deprivation. Motor neuron apoptosis resulting from both trophic factor deprivation and exposure to NRGbeta1 plus neurotrophins required the induction of neuronal nitric oxide synthase and peroxynitrite formation. Because motor neurons express both p75NTR and neuregulin erbB receptors during the period of embryonic programmed cell death, motor neuron survival may be the result of complex interactions between trophic and death factors, which may be the same molecules acting in different combinations.     

Cleavage of p75 neurotrophin receptor by alpha-secretase and gamma-secretase requires specific receptor domains

The p75 neurotrophin receptor (p75(NTR)), a member of the tumor necrosis factor superfamily of receptors, undergoes multiple proteolytic cleavage events. These events are initiated by an alpha-secretase-mediated release of the extracellular domain followed by a gamma-secretase-mediated intramembrane cleavage. However, the specific determinants of p75(NTR) cleavage events are unknown. Many other substrates of gamma-secretase cleavage have been identified, including Notch, amyloid precursor protein, and ErbB4, indicating there is broad substrate recognition by gamma-secretase. Using a series of deletion mutations and chimeric receptors of p75(NTR) and the related Fas receptor, we have identified domains that are essential for p75(NTR) proteolysis. The initial alpha-secretase cleavage was extracellular to the transmembrane domain. Unfortunately, deletion mutants were not capable of defining the requirements of ectodomain shedding. Although this cleavage is promiscuous with respect to amino acid sequence, its position with respect to the transmembrane domain is invariant. The generation of chimeric receptors exchanging different domains of noncleavable Fas receptor with p75(NTR), however, revealed that a discrete domain above the membrane is sufficient for efficient cleavage of p75(NTR). Mass spectrometric analysis confirmed the cleavage can occur with a truncated p75(NTR) displaying only 15 extracellular amino acids in the stalk region.