The human homologue of Caenorhabditis elegans CED-6 specifically promotes phagocytosis of apoptotic cells

A key feature of the process of programmed cell death (apoptosis) is the efficiency with which the dying cells are recognized and engulfed by phagocytes [1]. Apoptotic cells are rapidly cleared either by neighbouring cells acting as semi-professional phagocytes or by experts of the macrophage line, so that an inflammatory response is avoided [2]. The Caenorhabditis elegans gene ced-6 is required for efficient engulfment of apoptotic cells [3] and is one of a group of genes that define two partially redundant parallel pathways for the engulfment process [4] [5]. These pathways may be conserved across evolution, as two other engulfment genes have human homologues. A CED-5 homologue is part of a human CrkII-DOCK180-Rac signaling pathway proposed to mediate cytoskeletal reorganization [6] [7] [8] and a CED-7 homologue is similar to the ABC transporters [9] [10]. Here, we report the cloning and characterization of human CED-6, a human homologue of C. elegans CED-6. The 34 kDa hCED-6 protein is expressed in most tissues, some human cancer cells, and in primary human macrophages. We developed an assay that quantitates the phagocytic activity of mammalian macrophages: the number of apoptotic cells that have been internalized is measured by the uptake of lacZ-positive apoptotic cells by adherent transgenic macrophages. The results of this assay demonstrate that overexpression of hCED-6 promotes phagocytosis only of apoptotic cells and suggest that hCED-6 is the mammalian orthologue of C. elegans CED-6 and is a part of a highly conserved pathway that specifically mediates the phagocytosis of apoptotic cells.     

Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosis results from phospholipid scrambling and alters cell-surface morphology

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosylphosphorylcholine (C(6)-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca(2+) ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-beta-cyclodextrin, which is indicative of a loss of tight SM-cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C(6)- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.     

C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1

Externalization of phosphatidylserine, which is normally restricted to the inner leaflet of plasma membrane, is a hallmark of mammalian apoptosis. It is not known what activates and mediates the phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an annexin V-based phosphatidylserine labelling method and show that a majority of apoptotic germ cells in Caenorhabditis elegans have surface-exposed phosphatidylserine, indicating that phosphatidylserine externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the C. elegans apoptosis-inducing factor (AIF) homologue (WAH-1), a mitochondrial apoptogenic factor, or the C. elegans phospholipid scramblase 1 (SCRM-1), a plasma membrane protein, reduces phosphatidylserine exposure on the surface of apoptotic germ cells and compromises cell-corpse engulfment. WAH-1 associates with SCRM-1 and activates its phospholipid scrambling activity in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane phosphatidylserine externalization through its downstream effector, SCRM-1.     

Effects of RNA interference-mediated silencing of gamma-secretase complex components on cell sensitivity to caspase-3 activation

Familial Alzheimer's disease mutations in the presenilin 1 gene (PSEN1) have been previously shown to potentiate caspase activation and apoptosis in transfected cells and transgenic mice. However, the mechanism underlying this effect is not known. We set out to determine whether cellular sensitivity to caspase activation could be affected by modulating presenilin 1 (PS1) processing. PS1 processing was altered using RNA interference (RNAi) aimed at silencing the expression of the genes encoding the four components of the gamma-secretase complex, PSEN1, APH-1, PEN-2, and nicastrin. RNAi for these genes was carried out in naive H4 human neuroglioma cells, as well as H4 cell lines overexpressing either wild-type PSEN1 or the Familial Alzheimer's disease mutant PSEN1-Delta9 (PS1-mutant), that were induced to undergo apoptosis. In wild-type PSEN1 cells, RNAi for PEN-2, as expected, increased levels of full-length PS1 (PS1-FL) and decreased PS1 endoproteolysis. This was accompanied by potentiated caspase-3 activation in response to an apoptotic stimulus. In contrast, nicastrin RNAi, which only decreased levels of PS1-amino-terminal fragment and did not affect PS1-FL levels, had no effect on caspase-3 activation during apoptosis. Surprisingly, in the PS1-mutant cells, RNAi for PEN-2 (and APH-1) did not increase but instead reduced the levels of PS1-FL deleted for exon 9. In turn, this was accompanied by attenuated caspase-3 activation in response to an apoptotic stimulus. Finally, in naive H4 cells, PSEN1 RNAi also attenuated caspase-3 activation in response to an apoptotic stimulus. Collectively, these findings indicate that cellular sensitivity to caspase activation correlates with overall PS1 protein levels, particularly with levels of FL-PS1.     

Caenorhabditis elegans numb inhibits endocytic recycling by binding TAT-1 aminophospholipid translocase

Numb regulates endocytosis in many metazoans, but the mechanism by which it functions is not completely understood. Here we report that the Caenorhabditis elegans Numb ortholog, NUM-1A, a regulator of endocytic recycling, binds the C isoform of transbilayer amphipath transporter-1 (TAT-1), a P4 family adenosine triphosphatase and putative aminophospholipid translocase that is required for proper endocytic trafficking. We demonstrate that TAT-1 is differentially spliced during development and that TAT-1C-specific splicing occurs in the intestine where NUM-1A is known to function. NUM-1A and TAT-1C colocalize in vivo. We have mapped the binding site to an NXXF motif in TAT-1C. This motif is not required for TAT-1C function but is required for NUM-1A's ability to inhibit recycling. We demonstrate that num-1A and tat-1 defects are both suppressed by the loss of the activity of PSSY-1, a phosphatidylserine (PS) synthase. PS is mislocalized in intestinal cells with defects in tat-1 or num-1A function. We propose that NUM-1A inhibits recycling by inhibiting TAT-1C's ability to translocate PS across the membranes of recycling endosomes.     

Clearance of apoptotic cells in Caenorhabditis elegans

Programmed cell death, or apoptosis, is a genetically controlled process of cell suicide that is a common fate during an animal's life. In metazoans, apoptotic cells are rapidly removed from the body through the process of phagocytosis. Genetic analyses probing the mechanisms controlling the engulfment of apoptotic cells were pioneered in the nematode Caenorhabditis elegans. So far, at least seven genes have been identified that are required for the recognition and engulfment of apoptotic cells and have been shown to function in two partially redundant signaling pathways. Molecular characterization of their gene products has lead to the finding that similar genes act to control the same processes in other organisms, including mammals. In this paper, we review these exciting findings in C. elegans and discuss their implications in understanding the clearance of apoptotic cells in mammals.     

Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.

BACKGROUND: Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins. METHODS: We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis. RESULTS: Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content less, less than or approximately equal to 2.5%-8% for the membrane domains that stained with lactadherin but not annexin V. CONCLUSIONS: Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology.     

Phosphatidylserine exposure in living cells

Abstract

P4-ATPases, a subfamily of P-type ATPases, translocate cell membrane phospholipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet to generate and maintain membrane lipid asymmetry. Exposure of phosphatidylserine (PS) in the exoplasmic leaflet is well known to transduce critical signals for apoptotic cell clearance and platelet coagulation. PS exposure is also involved in many other biological processes, including myoblast and osteoclast fusion, and the immune response. Moreover, mounting evidence suggest that PS exposure is critical for neuronal regeneration and degeneration. In apoptotic cells, PS exposure is induced by irreversible activation of scramblases and inactivation of P4-ATPases. However, how PS is reversibly exposed and restored in viable cells during other biological processes remains poorly understood. In the present review, we discuss the physiological significance of reversible PS exposure in living cells, and the putative roles of flippases, floppases, and scramblases.     

Phosphatidylserine, a death knell

Virtually every cell in the body restricts phosphatidylserine (PS) to the inner leaflet of the plasma membrane by energy-dependent transport from the outer to the inner leaflet of the bilayer. Apoptotic cells of all types rapidly randomize the asymmetric distribution, bringing PS to the surface where it serves as a signal for phagocytosis. A myriad of phagocyte receptors have been implicated in the recognition of apoptotic cells, among them a PS receptor, yet few ligands other than PS have been identified on the apoptotic cell surface. Since apoptosis and the associated exposure of PS on the cell surface is probably over 600 million years old, it is not surprising that evolution has appropriated aspects of this process for specialized purposes such as blood coagulation, membrane fusion and erythrocyte differentiation. Failure to efficiently remove apoptotic cells may contribute to inflammatory responses and autoimmune diseases resulting from chronic, inappropriate exposure of PS.     

Hypoxic preconditioning requires the apoptosis protein CED-4 in C. elegans

Hypoxic preconditioning (HP) is a rapid and reversible proadaptive response to mild hypoxic exposure with such a response protecting cells from subsequent hypoxic or ischemic insult. HP mechanisms are of great interest because of their therapeutic potential and insight into metabolic adaptation and cell death. HP has been widely demonstrated in the vertebrate subphylum but not in invertebrates. Here, we report that the nematode Caenorhabditis elegans has a potent HP mechanism that protects the organism as well as its neurons and myocytes from hypoxic injury. The time course of C. elegans HP was consistent with vertebrate-delayed HP, appearing 16 hr after preconditioning and lasting at least 36 hr. The apoptosis pathway has been proposed as either a trigger or target of HP. Testing of mutations in the canonical C. elegans apoptosis pathway showed that in general, genes in this pathway are not required for HP. However, loss-of-function mutations in ced-4, which encodes an Apaf-1 homolog, completely blocked HP. RNAi silencing of ced-4 in adult animals immediately preceding preconditioning blocked HP, indicating that CED-4 is required in adults during or after preconditioning. CED-4/Apaf-1 is essential for HP in C. elegans and acts through a mechanism independent of the classical apoptosis pathway.     

Use of the monoclonal antibody DAKO-ERbeta (8D5-1) to measure oestrogen receptor beta in breast cancer cells

BACKGROUND: Following a lethal injury, two modes of cell death can be distinguished, apoptosis and primary necrosis. Cells pass through a prelethal stage characterized by a preservation of membrane integrity, in which they shrink (apoptosis) or swell (oncosis, the early phase of primary necrosis). During apoptosis, a loss of phospholipid asymmetry leads to exposure of phosphatidylserine (PS) residues on the outer leaflet of the plasma membrane. We examined whether the external PS exposure, initially supposed to be specific for apoptosis, was also observed in oncotic cells. METHODS: Human peripheral lymphocytes, Jurkat T cells, U937 cells, or HeLa cells were submitted to either apoptotic or oncotic stimuli. PS external exposure was assessed after binding of FITC-conjugated annexin V as was the loss of membrane integrity after propidium iodide (PI) uptake. Morphological examination was performed by optical or electron microscopy. RESULTS: Similarly to apoptotic cells, oncotic cells expose external PS residues while preserving membrane integrity. Consequently, oncotic cells exhibit the annexin V+ PI- phenotype, previously considered to be specific for apoptotic cells. CONCLUSIONS: This study concludes that the annexin V/PI assay does not discriminate between apoptosis and oncosis and that it can be a useful tool to study oncosis by flow cytometry.     

C1q binds phosphatidylserine and likely acts as a multiligand-bridging molecule in apoptotic cell recognition

Efficient apoptotic cell clearance is critical for maintenance of tissue homeostasis, and to control the immune responses mediated by phagocytes. Little is known about the molecules that contribute "eat me" signals on the apoptotic cell surface. C1q, the recognition unit of the C1 complex of complement, also senses altered structures from self and is a major actor of immune tolerance. HeLa cells were rendered apoptotic by UV-B treatment and a variety of cellular and molecular approaches were used to investigate the nature of the target(s) recognized by C1q. Using surface plasmon resonance, C1q binding was shown to occur at early stages of apoptosis and to involve recognition of a cell membrane component. C1q binding and phosphatidylserine (PS) exposure, as measured by annexin V labeling, proceeded concomitantly, and annexin V inhibited C1q binding in a dose-dependent manner. As shown by cosedimentation, surface plasmon resonance, and x-ray crystallographic analyses, C1q recognized PS specifically and avidly (K(D) = 3.7-7 x 10(-8) M), through multiple interactions between its globular domain and the phosphoserine group of PS. Confocal microscopy revealed that the majority of the C1q molecules were distributed in membrane patches where they colocalized with PS. In summary, PS is one of the C1q ligands on apoptotic cells, and C1q-PS interaction takes place at early stages of apoptosis, in newly organized membrane patches. Given its versatile recognition properties, these data suggest that C1q has the unique ability to sense different markers which collectively would provide strong eat me signals, thereby allowing efficient apoptotic cell removal.     

Nitrosative stress inhibits the aminophospholipid translocase resulting in phosphatidylserine externalization and macrophage engulfment: implications for the resolution of inflammation

Macrophage recognition of apoptotic cells depends on externalization of phosphatidylserine (PS), which is normally maintained within the cytosolic leaflet of the plasma membrane by aminophospholipid translocase (APLT). APLT is sensitive to redox modifications of its -SH groups. Because activated macrophages produce reactive oxygen and nitrogen species, we hypothesized that macrophages can directly participate in apoptotic cell clearance by S-nitrosylation/oxidation and inhibition of APLT causing PS externalization. Here we report that exposure of target HL-60 cells to nitrosative stress inhibited APLT, induced PS externalization, and enhanced recognition and elimination of "nitrosatively" modified cells by RAW 264.7 macrophages. Using S-nitroso-L-cysteine-ethyl ester (SNCEE) and S-nitrosoglutathione (GSNO) that cause intracellular and extracellular trans-nitrosylation of proteins, respectively, we found that SNCEE (but not GSNO) caused significant S-nitrosylation/oxidation of thiols in HL-60 cells. SNCEE also strongly inhibited APLT, activated scramblase, and caused PS externalization. However, SNCEE did not induce caspase activation or nuclear condensation/fragmentation suggesting that PS externalization was dissociated from the common apoptotic pathway. Dithiothreitol reversed SNCEE-induced S-nitrosylation, APLT inhibition, and PS externalization. SNCEE but not GSNO stimulated phagocytosis of HL-60 cells. Moreover, phagocytosis of target cells by lipopolysaccharide-stimulated macrophages was significantly suppressed by an NO. scavenger, DAF-2. Thus, macrophage-induced nitrosylation/oxidation plays an important role in cell clearance, and hence in the resolution of inflammation.     

AIF and Scythe (Bat3) Regulate Phosphatidylserine Exposure and Macrophage Clearance of Cells Undergoing Fas (APO-1)-Mediated Apoptosis

Phosphatidylserine (PS) exposure on the cell surface has been considered a characteristic feature of apoptosis and serves as a molecular cue for engulfment of dying cells by phagocytes. However, the mechanism of PS exposure is still not fully elucidated. Here we show that the cytosolic release from mitochondria of apoptosis-inducing factor (AIF) is required for PS exposure during death receptor-induced apoptosis and for efficient clearance of cell corpses by primary human macrophages. Fas-triggered PS exposure was significantly reduced upon siRNA-mediated silencing of AIF expression and by inhibition of the cytosolic translocation of AIF. In addition, AIF localizes to the plasma membrane upon Fas ligation and promotes activation of phospholipid scrambling activity. Finally, cytosolic stabilization of AIF through interaction with Scythe is shown to be involved in apoptotic PS exposure. Taken together, our results suggest an essential role for AIF and its binding partner Scythe in the pathway leading to apoptotic corpse clearance.     

Scythe cleavage during Fas (APO-1)-and staurosporine-mediated apoptosis

Scythe is a nuclear protein that has been implicated in the apoptotic process in Drosophila melanogaster; however, its role in apoptosis of mammalian cells is not fully elucidated. Here we show that cleavage of Scythe by caspase-3 occurs after activation of both the extrinsic (i.e. Fas/APO-1-mediated) and the intrinsic (i.e. staurosporine-induced) apoptosis pathway. Moreover, this caspase-dependent cleavage correlates with Scythe translocation from the nucleus to the cytosol. We also show that cytosolic re-localization of Scythe is required for Fas/APO-1-triggered phosphatidylserine (PS) exposure, a signal for macrophage clearance of apoptotic cells. Our data suggest that Scythe cleavage may represent a marker for caspase-3 activation and implicate cytosolic re-localization of Scythe in the pathway of PS exposure.     

Suicidal Membrane Repair Regulates Phosphatidylserine Externalization during Apoptosis

One of the hallmarks of apoptosis is the redistribution of phosphatidylserine (PS) from the inner-to-outer plasma membrane (PM) leaflet, where it functions as a ligand for phagocyte recognition and the suppression of inflammatory responses. The mechanism by which apoptotic cells externalize PS has been assumed to involve “scramblases” that randomize phospholipids across the PM bilayer. These putative activities, however, have not been unequivocally proven to be responsible for the redistribution of lipids. Because elevated cytosolic Ca²⁺ is critical to this process and is also required for activation of lysosome-PM fusion during membrane repair, we hypothesized that apoptosis could activate a “pseudo”-membrane repair response that results in the fusion of lysosomes with the PM. Using a membrane-specific probe that labels endosomes and lysosomes and fluorescein-labeled annexin 5 that labels PS, we show that the appearance of PS at the cell surface during apoptosis is dependent on the fusion of lysosomes with the PM, a process that is inhibited with the lysosomotrophe, chloroquine. We demonstrate that apoptotic cells evoke a persistent pseudo-membrane repair response that likely redistributes lysosomal-derived PS to the PM outer leaflet that leads to membrane expansion and the formation of apoptotic blebs. Our data suggest that inhibition of lysosome-PM fusion-dependent redistribution of PS that occurs as a result of chemotherapy- and radiotherapy-induced apoptosis will prevent PS-dependent anti-inflammatory responses that preclude the development of tumor- and patient-specific immune responses.There is increasing evidence that damaged plasma membranes (PM)² trigger an emergency Ca²⁺-dependent exocytotic repair response that patches the affected area by adding lysosome-derived membranes at the cell surface disruption site (1–5). Because high cytosolic Ca²⁺ concentrations trigger lysosome-PM fusion, the elevated cytosolic Ca²⁺ levels characteristic to apoptotic cells may also evoke a pseudo-repair mechanism that promotes lysosome-PM fusion. Indeed, similar to normal emergency repair responses, apoptosis is characterized by the appearance of organelle proteins and lipids at the PM surface (6–8). One critical distinction between the apoptotic and physiologic repair processes is the preservation of membrane lipid asymmetry. In normal cells, any perturbation in PS sidedness is corrected by restoration of basal cytosolic [Ca²⁺], reactivation of the Ca²⁺-inhibited aminophospholipid translocase (9, 10), and subsequent facilitated transport of PS back to the inner membrane leaflet of the cell. In apoptotic cells, however, persistent high cytosolic [Ca²⁺] precludes reactivation of the aminophospholipid translocase, and the redistributed PS remains in the outer membrane leaflet (11). The apparent similarities in these processes combined with observations that apoptotic cells express PS at the cell surface prompted us to investigate whether lysosome to PM fusion plays a role in the redistribution of PS during apoptosis.     

CD14-dependent clearance of apoptotic cells by human macrophages: the role of phosphatidylserine

Apoptotic-cell clearance is dependent on several macrophage surface molecules, including CD14. Phosphatidylserine (PS) becomes externalised during apoptosis and participates in the clearance process through its ability to bind to a novel receptor, PS-R. CD14 has the proven ability to bind phospholipids and may function as an alternative receptor for the externalised PS of apoptotic cells. Here we demonstrate that CD14 does not function preferentially as a PS receptor in apoptotic-cell clearance. Compared with phosphatidylcholine and phosphatidylethanolamine, PS was the least active phospholipid binding to human monocyte-derived macrophages and showed no specificity for soluble or membrane-anchored CD14. Significantly, PS-containing liposomes failed to inhibit CD14-dependent uptake of apoptotic cells by macrophages. PS exposure was, however, found to be insufficient for either CD14-dependent or CD14-independent apoptotic-cell uptake by phagocytes. The additional features that enable apoptotic-cell clearance are derived from mechanisms that can be divorced temporally from those responsible for the morphological features of apoptosis.     

Genome-wide RNAi identifies p53-dependent and -independent regulators of germ cell apoptosis in C. elegans

We used genome-wide RNA interference (RNAi) to identify genes that affect apoptosis in the C. elegans germ line. RNAi-mediated knockdown of 21 genes caused a moderate to strong increase in germ cell death. Genetic epistasis studies with these RNAi candidates showed that a large subset (16/21) requires p53 to activate germ cell apoptosis. Apoptosis following knockdown of the genes in the p53-dependent class also depended on a functional DNA damage response pathway, suggesting that these genes might function in DNA repair or to maintain genome integrity. As apoptotic pathways are conserved, orthologues of the worm germline apoptosis genes presented here could be involved in the maintenance of genomic stability, p53 activation, and fertility in mammals.