Decision making of the p53 network: Death by integration

The tumor suppressor protein p53 plays a central role in the multiple response pathways activated by DNA damage. In particular, p53 is involved in both the pro-survival response of cell cycle arrest and DNA repair, and the pro-death response of apoptosis. How does the p53 network coordinate the different pathways that lead to the opposite cell fates and what is its strategy in making the life-death decisions? To address these questions, we develop an integrated mathematical model that embraces three key modules of the p53 network: p53 core regulation, p53-induced cell cycle arrest and p53-dependent apoptosis initiation. Our analyses reveal that different aspects of the nuclear p53 dynamic profile are being used to differentially regulate the pro-survival and the pro-death modules. While the activation of the pro-survival module is dependent on the current or recent status of the DNA damage, the activation of the pro-death module relies on the accumulation or integration of the damage level over time. Thus, the cell will take the death fate if it cannot recover from the damage within a time period that is inversely proportional to the damage level. This “adaptive timer” strategy is likely to be adopted in other stress response systems.     

NR2A and NR2B subunits differentially mediate MAP kinase signaling and mitochondrial morphology following excitotoxic insult

NMDA receptors are essential for neurotransmission and key mediators of synaptic signaling, but they can also trigger deleterious degenerative processes that lead to cell death. Growing evidence suggests that selective blockade of the heterogeneous subunits that comprise the NMDA receptor may enable better control of pharmacotherapies for treating neurological diseases and injuries. We investigated the relationship between NMDAR activation, MAPK signaling, and mitochondrial shape following an excitotoxic insult. NR2A- and NR2B-containing NMDARs differentially mediated acute changes in cytosolic calcium, alterations in mitochondrial morphology, and phosphorylation of the MAPKs ERK and JNK. Activation of NR2A-containing NMDARs was associated with JNK phosphorylation that was neuroprotective in neuronal cultures subjected to excitotoxicity. In contrast, activation of NR2B-containing NMDARs triggered calcium accumulation in mitochondria that was strongly associated with mitochondrial swelling and neuronal cell death. Indeed, while blockade of NR2B-containing receptors was neuroprotective, this protection was lost when NR2A-initiated JNK phosphorylation was inhibited. Given the modest selectivity of the NR2A inhibitor, NVP-AAM077, the results highlight the significance of the relative, rather than absolute, activation of these two NMDA subtypes in modulating cell death pathways. Therefore, the balance between concurrent activation of NR2B-containing and NR2A-containing NMDARs dictates neuronal fate following excitotoxicity.     

Role of the proteolytic hierarchy between cathepsin L, cathepsin D and caspase-3 in regulation of cellular susceptibility to apoptosis and autophagy

The present investigation was undertaken to measure the relative abilities of pro-death versus pro-survival proteases in degrading each other and to determine how this might influence cellular susceptibility to death. For this, we first carried out in vitro experiments in which recombinant pro-death proteases (caspase-3 or cathepsin D) were incubated with the pro-survival protease (cathepsin L) in their respective optimal conditions and determined the effects of these reactions on enzyme integrity and activity. The results indicated that cathepsin L was able to degrade cathepsin D, which in turn cleaves caspase-3, however the later enzyme was unable to degrade any of the cathepsins. The consequences of this proteolytic sequence on cellular ability to undergo apoptosis or other types of cell death were studied in cells subjected to treatment with a specific inhibitor of cathepsin L or the corresponding siRNA. Both treatments resulted in suppression of cellular proliferation and the induction of a cell death with no detectable caspase-3 activation or DNA fragmentation, however, it was associated with increased accumulation of cathepsin D, cellular vaculolization, expression of the mannose-6-phosphate receptor, and the autophagy marker LC3-II, all of which are believed to be associated with autophagy. Genetic manipulations leading either to the gain or loss of cathepsin D expression implicated this enzyme as a key player in the switch from apoptosis to autophagy. Overall, these findings suggest that a hierarchy between pro-survival and pro-death proteases may have important consequences on cell fate.     

NMDA neuroprotection against a phosphatidylinositol-3 kinase inhibitor, LY294002 by NR2B-mediated suppression of glycogen synthase kinase-3beta-induced apoptosis

To identify the intracellular signaling pathways that mediate the pro-survival activity of NMDA receptors (NMDARs), we studied effects of exogenous NMDA on cultured rat cortical and hippocampal neurons that were treated with a phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002. NMDA at 5 or 10 microm protected against LY294002-induced apoptosis, suggesting NMDAR-mediated activation of a survival signaling pathway that is PI3K-independent. NR2B-specific NMDAR blockers antagonized anti-apoptotic effects of NMDA, indicating a critical role of NR2B NMDARs in the neuroprotection. NMDA at 10 microm suppressed LY294002-induced activation of a pro-apoptotic kinase, glycogen synthase kinase 3beta (GSK3beta). GSK3beta activation by LY294002 was associated with decreased levels of inhibitory GSK3beta phosphorylation at the Ser9 residue. However, NMDA did not prevent the LY294002-mediated decline of phospho-Ser9 levels. In addition, NMDA inhibited cortical neuron apoptosis induced by the overexpression of either wild type (wt) or Ser9Ala mutant form of GSK3beta, suggesting that NMDA suppressed GSK3beta in a Ser9-independent manner. Finally, inhibition of NR2B NMDARs reduced the NMDA protection against overexpression of GSK3betawt. These data indicate that moderate stimulation of NR2B NMDAR protects against inhibition of PI3K by a Ser9-independent inhibition of the pro-apoptotic activity of GSK3beta. Hence, the activation of NR2B and the Ser9-independent inhibition of GSK3beta are two newly identified elements of the signaling network that mediates the pro-survival effects of NMDA.     

Involvement of the GluN2A and GluN2B Subunits in Synaptic and Extrasynaptic N-methyl-d-aspartate Receptor Function and Neuronal Excitotoxicity

GluN2A and GluN2B are the major subunits of functional NMDA receptors (NMDAR). Previous studies have suggested that GluN2A and GluN2B may differentially mediate NMDAR function at synaptic and extrasynaptic locations and play opposing roles in excitotoxicity, such as neurodegeneration triggered by ischemic stroke and brain injury. By using pharmacological and molecular approaches to suppress or enhance the function of GluN2A and GluN2B in cultured cortical neurons, we examined NMDAR-mediated, bidirectional regulation of prosurvival signaling (i.e. the cAMP response element-binding protein (CREB)-Bdnf cascade) and cell death. Inhibition of GluN2A or GluN2B attenuated the up-regulation of prosurvival signaling triggered by the activation of either synaptic or extrasynaptic NMDAR. Inhibition of GluN2A or GluN2B also attenuated the down-regulation of prosurvival signaling triggered by the coactivation of synaptic and extrasynaptic receptors. The effects of GluN2B on CREB-Bdnf signaling were larger than those of GluN2A. Consistently, compared with suppression of GluN2A, suppression of GluN2B resulted in more reduction of NMDA- and oxygen glucose deprivation-induced excitotoxicity as well as NMDAR-mediated elevation of intracellular calcium. Moreover, excitotoxicity and down-regulation of CREB were exaggerated in neurons overexpressing GluN2A or GluN2B. Together, we found that GluN2A and GluN2B are involved in the function of both synaptic and extrasynaptic NMDAR, demonstrating that they play similar rather than opposing roles in NMDAR-mediated bidirectional regulation of prosurvival signaling and neuronal death.     

Inhibiting pro-death NMDA receptor signaling dependent on the NR2 PDZ ligand may not affect synaptic function or synaptic NMDA receptor signaling to gene expression

NMDA receptors (NMDARs) mediate ischemic brain damage, in part through interactions of the PDZ ligand of NR2 subunits with the PDZ domain proteins PSD-95 and neuronal nitric oxide synthase located within the NMDAR signaling complex. We have recently shown that this PDZ ligand-dependent pathway promotes neuronal death via p38 activation. A peptide mimetic of the NR2B PDZ ligand (TAT-NR2B9c) reduces p38-mediated death in vitro and p38-dependent ischemic damage in vivo. In the absence of the PDZ ligand-p38 pathway, such as in TAT-NR2B9c-treated neurons, or in NMDAR-expressing non-neuronal cells, NMDAR-dependent excitotoxicity is mediated largely by JNK and requires greater Ca2+ influx. A major reason for blocking pro-death signaling events downstream of the NMDAR as an anti-excitotoxic strategy is that it may spare physiological synaptic function and signaling. We find that neuroprotective doses of TAT-NR2B9c do not alter the frequency of spontaneous synaptic events within networks of cultured cortical neurons nor is mini-EPSC frequency altered. Furthermore, TAT-NR2B9c does not inhibit the capacity of synaptic NMDAR activity to promote neuroprotective changes in gene expression, including the up-regulation of PACAP via CREB, and suppression of the pro-oxidative FOXO target gene Txnip. Thus, while the NR2 PDZ ligand does not account for all the excitotoxic effects of excessive NMDAR activity, these findings underline the value of the specific targeting of death pathways downstream of the NMDAR.     

Regulating RIPK1: another way in which ULK1 contributes to survival

ABSTRACT

The mammalian ULK1 is the central initiating kinase of bulk and selective macroautophagy/autophagy processes. In the past, both autophagy-relevant and non-autophagy-relevant substrates of this Ser/Thr kinase have been reported. Here, we describe our recent finding that ULK1 also regulates TNF signaling pathways. We find that inhibition of autophagy or specifically ULK1 increases TNF-induced cell death. This autophagy-independent pro-survival function of ULK1 is mediated via the phosphorylation of RIPK1 at Ser357. RIPK1 is the central mediator of pro-inflammatory or pro-death signaling pathways induced by TNF, and ULK1-dependent phosphorylation regulates RIPK1 activation and distribution to different intracellular signaling complexes. Our results indicate that ULK1 exerts a cyto-protective function not only by initiating autophagy, but also by controlling RIPK1-mediated cell death.     

Cytosolic accumulation of HSP60 during apoptosis with or without apparent mitochondrial release: evidence that its pro-apoptotic or pro-survival functions involve differential interactions with caspase-3

Most heat shock proteins (HSPs) have pro-survival functions. However, the role of HSP60, a mitochondrial matrix protein, is somewhat controversial with both pro-survival and pro-apoptotic functions reported. Here we show that in numerous apoptotic systems HSP60 protein accumulates in the cytosol. In BMD188-induced cell death, HSP60 accumulates in the cytosol with significant mitochondrial release. In contrast, in apoptosis induced by multiple other inducers, the cytosolic HSP60 accumulates without an apparent mitochondrial release. The short interfering RNA-mediated knockdown experiments revealed that in BMD188-induced apoptosis, HSP60 has a pro-death function and that the pro-death role of HSP60 seems to involve caspase-3 maturation and activation in the cytosol. In contrast, HSP60 appears to play a pro-survival role in other apoptotic systems where there is no apparent mitochondrial release as its knockdown promotes cell death. In these latter apoptotic systems HSP60 does not associate with active caspase-3. In both cases, HSP60 does not appreciably interact with Bax. Taken together, our results suggest the following: 1) cytosolic accumulation of HSP60 represents a common phenomenon during apoptosis induction; 2) cytosolic HSP60 accumulation during apoptosis occurs either with or without apparent mitochondrial release; and 3) the cytosolically accumulated HSP60 possesses either pro-survival or pro-death functions, which involves differential interactions with caspase-3.     

Redox modulation by S-nitrosylation contributes to protein misfolding, mitochondrial dynamics, and neuronal synaptic damage in neurodegenerative diseases

The pathological processes of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases engender synaptic and neuronal cell damage. While mild oxidative and nitrosative (nitric oxide (NO)-related) stress mediates normal neuronal signaling, excessive accumulation of these free radicals is linked to neuronal cell injury or death. In neurons, N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation and subsequent Ca(2+) influx can induce the generation of NO via neuronal NO synthase. Emerging evidence has demonstrated that S-nitrosylation, representing covalent reaction of an NO group with a critical protein thiol, mediates the vast majority of NO signaling. Analogous to phosphorylation and other posttranslational modifications, S-nitrosylation can regulate the biological activity of many proteins. Here, we discuss recent studies that implicate neuropathogenic roles of S-nitrosylation in protein misfolding, mitochondrial dysfunction, synaptic injury, and eventual neuronal loss. Among a growing number of S-nitrosylated proteins that contribute to disease pathogenesis, in this review we focus on S-nitrosylated protein-disulfide isomerase (forming SNO-PDI) and dynamin-related protein 1 (forming SNO-Drp1). Furthermore, we describe drugs, such as memantine and newer derivatives of this compound that can prevent both hyperactivation of extrasynaptic NMDARs as well as downstream pathways that lead to nitrosative stress, synaptic damage, and neuronal loss.     

To be or not to be, the level of autophagy is the question: dual roles of autophagy in the survival response to starvation

Autophagy is an evolutionally conserved lysosomal pathway used to degrade and turn over long-lived proteins and cytoplasmic organelles. Since autophagy was discovered, it has been thought to act as a pro-survival response to several stresses, especially starvation, at the cell and organism levels by providing recycled metabolic substrates to maintain energy homeostasis. However, several recent studies suggest that autophagy also plays a pro-death role through an autophagic cell death pathway mostly at the cellular level. The mechanism by which autophagy could perform these seemingly opposite roles as a pro-survival and a pro-death mechanism remained elusive until recently. Using C. elegans as a model system, we found that physiological levels of autophagy promote optimal survival of C. elegans during starvation, but either insufficient or excessive levels of autophagy render C. elegans starvation-hypersensitive. Furthermore, we found that muscarinic acetylcholine receptor signaling is important in modulating the level of autophagy during starvation, perhaps through DAP kinase and RGS-2. Our recent study provides in vivo evidence that levels of autophagy are critical in deciding its promotion of either survival or death: Physiological levels of autophagy are pro-survival, whereas insufficient or excessive levels of autophagy are pro-death.     

Endoplasmic reticulum-targeting photosensitizer Hypericin confers chemo-sensitization towards oxaliplatin through inducing pro-death autophagy

Hypericin is an endoplasmic reticulum (ER)-located photosensitizer, which causes oxidative damage to ER during photodynamic therapy (PDT). Hypericin-mediated PDT (HY-PDT) has been confirmed to enhance chemo-sensitivity of oxaliplatin (L-OHP) in colon cancer cells. The present study reveals that autophagy plays a key role in chemosensitization during HY-PDT. We proved pro-death autophagy was required for sensitization and HY-PDT/L-OHP antitumor synergism. High dosage of HY-PDT induced autophagic cell death; while low dose of HY-PDT predominantly triggered protective autophagy and promoted cell proliferation. Low dose of HY-PDT reduced the cytotoxicity of L-OHP in oxaliplatin-resistant colon cancer cells. Different level of autophagy therefore contributed to the opposite effect of HY-PDT on cell fate and chemo-sensitivity. Furthermore, we revealed the role of CHOP as a regulator connecting pro-survival and pro-death autophagy under ER damage. High dose of HY-PDT induced massive ROS generation and severe ER stress, which then led to induction of CHOP. CHOP thereby activated CHOP/TRIB3/Akt/mTOR cascade and triggered autophagic cell death. Additionally, when apoptotic pathway was blocked, cells treated with high dose of HY-PDT preferentially underwent death through autophagic pathway. On the other hand, suppression of autophagy made cells more vulnerable to apoptosis under low dose of HY-PDT. These results provided new evidences for the clinical application of ER-targeting PDT in modifying chemosensitivity of colorectal cancer therapy.     

Potentiation of NMDA Receptor-Dependent Cell Responses by Extracellular High Mobility Group Box 1 Protein

Background

Extracellular high mobility group box 1 (HMGB1) protein can operate in a synergistic fashion with different signal molecules promoting an increase of cell Ca²⁺ influx. However, the mechanisms responsible for this effect of HMGB1 are still unknown.

Principal Findings

Here we demonstrate that, at concentrations of agonist per se ineffective, HMGB1 potentiates the activation of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR) in isolated hippocampal nerve terminals and in a neuroblastoma cell line. This effect was abolished by the NMDA channel blocker MK-801. The HMGB1-facilitated NMDAR opening was followed by activation of the Ca²⁺-dependent enzymes calpain and nitric oxide synthase in neuroblastoma cells, resulting in an increased production of NO, a consequent enhanced cell motility, and onset of morphological differentiation. We have also identified NMDAR as the mediator of HMGB1-stimulated murine erythroleukemia cell differentiation, induced by hexamethylenebisacetamide. The potentiation of NMDAR activation involved a peptide of HMGB1 located in the B box at the amino acids 130–139. This HMGB1 fragment did not overlap with binding sites for other cell surface receptors of HMGB1, such as the advanced glycation end products or the Toll-like receptor 4. Moreover, in a competition assay, the HMGB1_(130–139) peptide displaced the NMDAR/HMGB1 interaction, suggesting that it comprised the molecular and functional site of HMGB1 regulating the NMDA receptor complex.

Conclusion

We propose that the multifunctional cytokine-like molecule HMGB1 released by activated, stressed, and damaged or necrotic cells can facilitate NMDAR-mediated cell responses, both in the central nervous system and in peripheral tissues, independently of other known cell surface receptors for HMGB1.     

Timing the multiple cell death pathways initiated by Rose Bengal acetate photodynamic therapy

Rose Bengal acetate photodynamic therapy (RBAc–PDT) induced multiple cell death pathways in HeLa cells through ROS and ER stress. Indeed, apoptosis was the first preferred mechanism of death, and it was triggered by at least four different pathways, whose independent temporal activation ensures cell killing when one or several of the pathways are inactivated. Apoptosis occurred as early as 1 h after PDT through activation of intrinsic pathways, followed by activation of extrinsic, caspase-12-dependent and caspase-independent pathways, and by autophagy. The onset of the different apoptotic pathways and autophagy, that in our system had a pro-death role, was timed by determining the levels of caspases 9, 8, 3 and 12; Bcl-2 family; Hsp70; LC3B; GRP78 and phospho-eIF2α proteins. Interestingly, inhibition of one pathway, that is, caspase-9 (Z-LEHD-FMK), caspase-8 (Z-IETD-FMK), pan-caspases (Z-VAD-FMK), autophagy (3-MA) and necrosis (Nec-1), did not impair the activation of the others, suggesting that the independent onset of the different apoptotic pathways and autophagy did not occur in a subordinated manner. Altogether, our data indicate RBAc as a powerful photosensitiser that induces a prolonged cytotoxicity and time-related cell death onset by signals originating from or converging on almost all intracellular organelles. The fact that cancer cells can die through different mechanisms is a relevant clue in the choice and design of anticancer PDT.     

Over-activation of NMDA receptors promotes ABCA1 degradation and foam cell formation

ATP-binding cassette transporter A1 (ABCA1) is an essential regulator of intracellular cholesterol efflux. Secreted cholesterol binds to lipid-free apolipoprotein A-I (apoA-I) in peripheral blood to constitute high-density lipoprotein cholesterol (HDL) complexes. ABCA1 protein on the surface of macrophages acts as a crucial controller in preventing cholesterol accumulation. Importantly, ABCA1 is unstable and easily degraded via a series of biochemical activities, including but not limited to calpain-mediated and ubiquitin-proteasome system-mediated processes. How accelerated ABCA1 degradation impacts disordered lipid metabolism in macrophages and foam cell formation is unclear. N-methyl d-aspartate receptors (NMDARs) are ionotropic glutamate receptors with high calcium permeability. Calcium influx via NMDARs activates downstream signaling pathways. Over-activation of NMDARs stimulated by NMDA contributes to dysfunctional lipid metabolism in macrophages and foam cell formation via promotion of calpain-mediated ABCA1 proteolysis. However, increased NMDAR activity does not affect liver X receptor expression or ABCA1 mRNA levels. Following NMDA receptor silencing or calpain inhibition, NMDA treatment did not reduce ABCA1 protein levels, nor caused lipid accumulation in macrophages. In addition, NMDAR over-activation activates NF-κB signaling to promote IL-1β and IL-6 macrophage marker expression. However, NMDAR silencing and calpain inhibition reduce inflammatory macrophage responses. In summary, our study suggests that NMDAR activation reduces surface ABCA1 protein, promotes lipid accumulation, and induces the production and secretion of many inflammatory mediators in macrophages, possibly through enhanced calpain-mediated ABCA1 protein degradation. Thus, the NMDAR receptor may be a novel pharmacologic target for atherosclerosis therapy.     

RIP1 comes back to life as a cell death regulator in TNFR1 signaling

Cell death induction by tumor necrosis factor has been an intensively studied area for the last two decades. Although it may appear that the skeleton should have been picked clean by now, new secrets about tumor necrosis factor death signaling are still being uncovered. In particular, the recent evidence that ubiquitination of the death kinase receptor-interacting protein 1 regulates its participation in apoptotic and necrotic cell death is opening up unexplored avenues in the catacombs of tumor necrosis factor death signaling. In this minireview, we focus on two major cell-death checkpoints that determine whether receptor-interacting protein 1 functions as a pro-survival or pro-death molecule.