Advances in apoptosis research

Apoptosis, also called programmed cell death, has attracted great attention in recent years. After its discovery by Carl Vogt in 1842, apoptosis research was dormant for more than a century. Its rediscovery in the second half of this century, and the coining of the term apoptosis in 1972 by Kerr, Wyllie, and Currie, ignited an unparalleled interest in this field of science. The number of publications related to apoptosis has been growing exponentially every year ever since. This is mainly due to three major advances, two of which have been made recently and one that is currently seen. First, studies with the small nematode Caenorhabditis elegans have identified a number of apoptosis regulating genes—the first evidence that cell death is an active process under genetic control. Many of these genes have mammalian homologs that, like their worm counterparts, seem to regulate mammalian apoptosis. Second, elucidation of the signal transduction pathways of apoptosis has lead especially to the identification of specific death signaling molecules such as a new family of cysteine proteases, the caspases. Third, it has now become clear that many diseases are characterized by dysregulation of apoptotic programs. Many of these programs involve a family of receptors and their ligands, the death receptor/ligand family. The hope now is to interfere with apoptosis regulation in these systems and to develop new therapeutic concepts.

Function of the oxidative burst in hypersensitive disease resistance.

Microbial elicitors or attempted infection with an avirulent pathogen strain causes the rapid production of reactive oxygen intermediates. Recent findings indicate that H2O2 from this oxidative burst plays a central role in the orchestration of the hypersensitive response: (i) as the substrate driving the cross-linking of cell wall structural proteins to slow microbial ingress prior to the deployment of transcription-dependent defenses and to trap pathogens in cells destined to undergo hypersensitive cell death, (ii) as a local threshold trigger of this programmed death in challenged cells, and (iii) as a diffusible signal for the induction in adjacent cells of genes encoding cellular protectants such as glutathione S-transferase and glutathione peroxidase. These findings provide the basis for an integrated model for the orchestration of the localized hypersensitive resistance response to attack by an avirulent pathogen.

Human immunodeficiency virus 1 envelope-initiated G2-phase programmed cell death.

Despite intensive investigation, no clearly defined mechanism explaining human immunodeficiency virus (HIV)-induced cell killing has emerged. HIV-1 infection is initiated through a high-affinity interaction between the HIV-1 external envelope glycoprotein (gp120) and the CD4 receptor on T cells. Cell killing is a later event intimately linked by in vitro genetic analyses with the fusogenic properties of the HIV envelope glycoprotein gp120 and transmembrane glycoprotein gp41. In this report, we describe aberrancies in cell cycle regulatory proteins initiated by cell-cell contact between T cells expressing HIV-1 envelope glycoproteins and other T cells expressing CD4 receptors. Cells rapidly accumulate cyclin B protein and tyrosine-hyperphosphorylated p34cdc2 (cdk1) kinase, indicative of cell cycle arrest at G2 phase. Moreover, these cells continue to synthesize cyclin B protein, enlarge and display an abnormal ballooned morphology, and disappear from the cultures in a pattern previously described for cytotoxicity induced by DNA synthesis (S phase) inhibitors. Similar changes are observed in peripheral blood mononuclear cells infected in vitro with pathogenic primary isolates of HIV-1.

The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an LFA-1 integrin ligand

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-1). However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2-Å resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus–ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the insertion (I)-domain on the α chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.

Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: Implications for AIDS pathology

The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal fluid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of fluorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potential-sensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal fluid.

Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1α, a potent ligand for the HIV-1 “fusin” coreceptor

Stromal cell-derived factor-1α (SDF-1α ) is a member of the chemokine superfamily and functions as a growth factor and chemoattractant through activation of CXCR4/LESTR/Fusin, a G protein-coupled receptor. This receptor also functions as a coreceptor for T-tropic syncytium-inducing strains of HIV-1. SDF-1α antagonizes infectivity of these strains by competing with gp120 for binding to the receptor. The crystal structure of a variant SDF-1α ([N33A]SDF-1α ) prepared by total chemical synthesis has been refined to 2.2-Å resolution. Although SDF-1α adopts a typical chemokine β-β-β-α topology, the packing of the α-helix against the β-sheet is strikingly different. Comparison of SDF-1α with other chemokine structures confirms the hypothesis that SDF-1α may be either an ancestral protein from which all other chemokines evolved or the chemokine that is the least divergent from a primordial chemokine. The structure of SDF-1α reveals a positively charged surface ideal for binding to the negatively charged extracellular loops of the CXCR4 HIV-1 coreceptor. This ionic complementarity is likely to promote the interaction of the mobile N-terminal segment of SDF-1α with interhelical sites of the receptor, resulting in a biological response.

CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4+ T cells

AIDS is characterized by a progressive decrease of CD4+ helper T lymphocytes. Destruction of these cells may involve programmed cell death, apoptosis. It has previously been reported that apoptosis can be induced even in noninfected cells by HIV-1 gp120 and anti-gp120 antibodies. HIV-1 gp120 binds to T cells via CD4 and the chemokine coreceptor CXCR4 (fusin/LESTR). Therefore, we investigated whether CD4 and CXCR4 mediate gp120-induced apoptosis. We used human peripheral blood lymphocytes, malignant T cells, and CD4/CXCR4 transfectants, and found cell death induced by both cell surface receptors, CD4 and CXCR4. The induced cell death was rapid, independent of known caspases, and lacking oligonucleosomal DNA fragmentation. In addition, the death signals were not propagated via p56lck and Giα. However, the cells showed chromatin condensation, morphological shrinkage, membrane inversion, and reduced mitochondrial transmembrane potential indicative of apoptosis. Significantly, apoptosis was exclusively observed in CD4+ but not in CD8+ T cells, and apoptosis triggered via CXCR4 was inhibited by stromal cell-derived factor-1, the natural CXCR4 ligand. Thus, this mechanism of apoptosis might contribute to T cell depletion in AIDS and might have major implications for therapeutic intervention.

The role of polyamine catabolism in polyamine analogue-induced programmed cell death

N1-ethyl-N11-[(cyclopropyl)methyl]-4,8,-diazaundecane (CPENSpm) is a polyamine analogue that represents a new class of antitumor agents that demonstrate phenotype-specific cytotoxic activity. However, the precise mechanism of its selective cytotoxic activity is not known. CPENSpm treatment results in the superinduction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT) in sensitive cell types and has been demonstrated to induce programmed cell death (PCD). The catalysis of polyamines by the SSAT/polyamine oxidase (PAO) pathway produces H2O2 as one product, suggesting that PCD produced by CPENSpm may be, in part, due to oxidative stress as a result of H2O2 production. In the sensitive human nonsmall cell line H157, the coaddition of catalase significantly reduces high molecular weight (HMW) DNA (≥50 kb) and nuclear fragmentation. Important to note, specific inhibition of PAO by N,N′-bis(2,3-butadienyl)-1,4-butane-diamine results in a significant reduction of the formation of HMW DNA and nuclear fragmentation. In contrast, the coaddition of catalase or PAO inhibitor has no effect on reducing HMW DNA fragmentation induced by N1-ethyl-N11-[(cycloheptyl)methyl]-4,8,-diazaundecane, which does not induce SSAT and does not deplete intracellular polyamines. These results strongly suggest that H2O2 production by PAO has a role in CPENSpm cytotoxicity in sensitive cells via PCD and demonstrate a potential basis for differential sensitivity to this promising new class of antineoplastic agents. Furthermore, the data suggest a general mechanism by which, under certain stimuli, cells can commit suicide through catabolism of the ubiquitous intracellular polyamines.

Multiple loop structures critical for ligand binding of the integrin α4 subunit in the upper face of the β-propeller mode 1

A non-I-domain integrin, α4β1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of α4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of α4 (which spans repeats 2–5 of the seven N-terminal repeats) with the corresponding regions of α5. Swapping residues 112–131 in repeat 2, or residues 237–247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40–52 in repeat 1, residues 151–164 in repeat 3, or residues 282–288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112–131, 151–164, and 186–191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published β-propeller folding model of the integrin α4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65–72], in which seven four-stranded β-sheets are arranged in a torus around a pseudosymmetric axis. The regions of α4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the β-propeller model, although they are not adjacent in the primary structure.

Tyrosine replacement in P-selectin glycoprotein ligand-1 affects distinct kinetic and mechanical properties of bonds with P- and L-selectin

Selectins are adhesion molecules that initiate tethering and rolling of leukocytes on the vessel wall. Rolling requires rapid formation and breakage of selectin–ligand bonds that must have mechanical strength to resist premature dissociation by the forces applied in shear flow. P- and L-selectin bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1), a mucin on leukocytes. To define determinants on PSGL-1 that contribute to the kinetic and mechanical properties of bonds with selectins, we compared rolling of transfected preB cells expressing P- or L-selectin on transfected cell monolayers expressing wild-type PSGL-1 or PSGL-1 constructs with substitutions in targeted N-terminal residues. Rolling through P- or L-selectin required a Thr or Ser at a specific position on PSGL-1, the attachment site for an essential O-glycan, but required only one of three nearby Tyr residues, which are sites for Tyr-SO3 formation. The adhesive strengths and numbers of cells rolling through P- or L-selectin were similar on wild-type PSGL-1 and on each of the three PSGL-1 constructs containing only a single Tyr. However, the cells rolled more irregularly on the single-Tyr forms of PSGL-1. Analysis of the lifetimes of transient tethers on limiting densities of PSGL-1 revealed that L-selectin dissociated faster from single-Tyr than wild-type PSGL-1 at all shears examined. In sharp contrast, P-selectin dissociated faster from single-Tyr than wild-type PSGL-1 at higher shear but not at lower shear. Thus, tyrosine replacements in PSGL-1 affect distinct kinetic and mechanical properties of bonds with P- and L-selectin.

The α1 domain of HLA-G1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: Is HLA-G the public ligand for natural killer cell inhibitory receptors?

We have investigated the protective role of the membrane-bound HLA-G1 and HLA-G2 isoforms against natural killer (NK) cell cytotoxicity. For this purpose, HLA-G1 and HLA-G2 cDNAs were transfected into the HLA class I-negative human K562 cell line, a known reference target for NK lysis. The HLA-G1 protein, encoded by a full-length mRNA, presents a structure similar to that of classical HLA class I antigens. The HLA-G2 protein, deduced from an alternatively spliced transcript, consists of the α1 domain linked to the α3 domain. In this study we demonstrate that (i) HLA-G2 is present at the cell surface as a truncated class I molecule associated with β2-microglobulin; (ii) NK cytolysis, observed in peripheral blood mononuclear cells and in polyclonal CD3− CD16+ CD56+ NK cells obtained from 20 donors, is inhibited by both HLA-G1 and HLA-G2; this HLA-G-mediated inhibition is reversed by blocking HLA-G with a specific mAb; this led us to the conjecture that HLA-G is the public ligand for NK inhibitory receptors (NKIR) present in all individuals; (iii) the α1 domain common to HLA-G1 and HLA-G2 could mediate this protection from NK lysis; and (iv) when transfected into the K562 cell line, both HLA-G1 and HLA-G2 abolish lysis by the T cell leukemia NK-like YT2C2 clone due to interaction between the HLA-G isoform on the target cell surface and a membrane receptor on YT2C2. Because NKIR1 and NKIR2, known to interact with HLA-G, were undetectable on YT2C2, we conclude that a yet-unknown specific receptor for HLA-G1 and HLA-G2 is present on these cells.

The Epstein–Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-κB

The Epstein–Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model that LMP1 is a constitutively activated receptor that transduces signals for transformation through its carboxyl-terminal cytoplasmic tail. One transformation effector site (TES1), located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD was the only protein that interacted with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated with wild-type LMP1 but not with isoleucine-mutated LMP1 in mammalian cells, and TRADD constitutively associated with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediated high-level NF-κB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-κB activation also appears to be a critical component of long-term outgrowth.

NF-κB and AP-1 Activation by Nitric Oxide Attenuated Apoptotic Cell Death in RAW 264.7 Macrophages

A toxic dose of the nitric oxide (NO) donor S-nitrosoglutathione (GSNO; 1 mM) promoted apoptotic cell death of RAW 264.7 macrophages, which was attenuated by cellular preactivation with a nontoxic dose of GSNO (200 μM) or with lipopolysaccharide, interferon-γ, and NG-monomethyl-l-arginine (LPS/IFN-γ/NMMA) for 15 h. Protection from apoptosis was achieved by expression of cyclooxygenase-2 (Cox-2). Here we investigated the underlying mechanisms leading to Cox-2 expression. LPS/IFN-γ/NMMA prestimulation activated nuclear factor (NF)-κB and promoted Cox-2 expression. Cox-2 induction by low-dose GSNO demanded activation of both NF-κB and activator protein-1 (AP-1). NF-κB supershift analysis implied an active p50/p65 heterodimer, and a luciferase reporter construct, containing four copies of the NF-κB site derived from the murine Cox-2 promoter, confirmed NF-κB activation after NO addition. An NF-κB decoy approach abrogated not only Cox-2 expression after low-dose NO or after LPS/IFN-γ/NMMA but also inducible protection. The importance of AP-1 for Cox-2 expression and cell protection by low-level NO was substantiated by using the extracellular signal-regulated kinase inhibitor PD98059, blocking NO-elicited Cox-2 expression, but leaving the cytokine signal unaltered. Transient transfection of a dominant-negative c-Jun mutant further attenuated Cox-2 expression by low-level NO. Whereas cytokine-mediated Cox-2 induction relies on NF-κB activation, a low-level NO–elicited Cox-2 response required activation of both NF-κB and AP-1.

Ligand-induced Trafficking of the Sphingosine-1-phosphate Receptor EDG-1

The endothelial-derived G-protein–coupled receptor EDG-1 is a high-affinity receptor for the bioactive lipid mediator sphingosine-1-phosphate (SPP). In the present study, we constructed the EDG-1–green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP–EDG-1 interaction. SPP binds to EDG-1–GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-1–GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a τ1/2 of ∼15 min. The EDG-1–GFP–containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-1-phosphate, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-1–GFP recycles back to the plasma membrane with a τ1/2 of ∼30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-1 via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process.

Identification of Osteopontin as a Novel Ligand for the Integrin α8β1 and Potential Roles for This Integrin–Ligand Interaction in Kidney Morphogenesis

Epithelio–mesenchymal interactions during kidney organogenesis are disrupted in integrin α8β1-deficient mice. However, the known ligands for integrin α8β1—fibronectin, vitronectin, and tenascin-C—are not appropriately localized to mediate all α8β1 functions in the kidney. Using a method of general utility for determining the distribution of unknown integrin ligands in situ and biochemical characterization of these ligands, we identified osteopontin (OPN) as a ligand for α8β1. We have coexpressed the extracellular domains of the mouse α8 and β1 integrin subunits as a soluble heterodimer with one subunit fused to alkaline phosphatase (AP) and have used the α8β1-AP chimera as a histochemical reagent on sections of mouse embryos. Ligand localization with α8β1-AP in developing bone and kidney was observed to be overlapping with the distribution of OPN. In “far Western” blots of mouse embryonic protein extracts, bands were detected with sizes corresponding to fibronectin, vitronectin, and unknown proteins, one of which was identical to the size of OPN. In a solid-phase binding assay we demonstrated that purified OPN binds specifically to α8β1-AP. Cell adhesion assays using K562 cells expressing α8β1 were used to confirm this result. Together with a recent report that anti-OPN antibodies disrupt kidney morphogenesis, our results suggest that interactions between OPN and integrin α8β1 may help regulate kidney development and other morphogenetic processes.