Lorraine A. O'Reilly

Bim: a novel member of the Bcl‐2 family that promotes apoptosis

Certain members of the Bcl‐2 family inhibit apoptosis while others facilitate this physiological process of cell death. An expression screen for proteins that bind to Bcl‐2 yielded a small novel protein, denoted Bim, whose only similarity to any known protein is the short (nine amino acid) BH3 motif shared by most Bcl‐2 homologues. Bim provokes apoptosis, and the BH3 region is required for Bcl‐2 binding and for most of its cytotoxicity. Like Bcl‐2, Bim possesses a hydrophobic C‐terminus and localizes to intracytoplasmic membranes. Three Bim isoforms, probably generated by alternative splicing, all induce apoptosis, the shortest being the most potent. Wild‐type Bcl‐2 associates with Bim in vivo and modulates its death function, whereas Bcl‐2 mutants that lack survival function do neither. Significantly, Bcl‐xL and Bcl‐w, the two closest homologues of Bcl‐2, also bind to Bim and inhibit its activity, but more distant viral homologues, adenovirus E1B19K and Epstein–Barr virus BHRF‐1, can do neither. Hence, Bim appears to act as a ‘death ligand’ which can only neutralize certain members of the pro‐survival Bcl‐2 sub‐family.

ER Stress Triggers Apoptosis by Activating BH3-Only Protein Bim

Endoplasmic reticulum (ER) stress caused by misfolded proteins or cytotoxic drugs can kill cells and although activation of this pathway has been implicated in the etiology of certain degenerative disorders its mechanism remains unresolved. Bim, a proapoptotic BH3-only member of the Bcl-2 family is required for initiation of apoptosis induced by cytokine deprivation or certain stress stimuli. Its proapoptotic activity can be regulated by several transcriptional or posttranslational mechanisms, such as ERK-mediated phosphorylation, promoting its ubiquitination and proteasomal degradation. We found that Bim is essential for ER stress-induced apoptosis in a diverse range of cell types both in culture and within the whole animal. ER stress activates Bim through two novel pathways, involving protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and proteasomal degradation and CHOP-C/EBPalpha-mediated direct transcriptional induction. These results define the molecular mechanisms of ER stress-induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases.

Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1-/- mice.

Signals from cytokine and antigen receptors play crucial roles during lymphocyte development. Mice lacking interleukin-7 receptor are lymphopenic, due to a defect in cell expansion at an early stage of differentiation, and the few mature T cells that develop in IL-7R-/- animals are functionally impaired. Both defects were rescued completely by overexpression of the anti-apoptosis protein Bcl-2. T cell progenitors lacking antigen receptor molecules are also blocked in differentiation and die, presumably because they fail to receive a positive signal via their pre-T cell receptor. Surprisingly, Bcl-2 did not promote survival or differentiation of T cells in rag-1-/- mice. These results provide evidence that blocking apoptosis is the essential function of IL-7R during differentiation and activation of T lymphocytes and that pre-TCR signaling blocks a pathway to apoptosis that is insensitive to Bcl-2.

Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome

Apoptosis is an evolutionarily conserved cell suicide process executed by cysteine proteases (caspases) and regulated by the opposing factions of the Bcl-2 protein family. Mammalian caspase-9 and its activator Apaf-1 were thought to be essential, because mice lacking either of them display neuronal hyperplasia and their lymphocytes and fibroblasts seem resistant to certain apoptotic stimuli. Because Apaf-1 requires cytochrome c to activate caspase-9, and Bcl-2 prevents mitochondrial cytochrome c release, Bcl-2 is widely believed to inhibit apoptosis by safeguarding mitochondrial membrane integrity. Our results suggest a different, broader role, because Bcl-2 overexpression increased lymphocyte numbers in mice and inhibited many apoptotic stimuli, but the absence of Apaf-1 or caspase-9 did not. Caspase activity was still discernible in cells lacking Apaf-1 or caspase-9, and a potent caspase antagonist both inhibited apoptosis and retarded cytochrome c release. We conclude that Bcl-2 regulates a caspase activation programme independently of the cytochrome c/Apaf-1/caspase-9 ‘apoptosome’, which seems to amplify rather than initiate the caspase cascade.

THE CELL DEATH INHIBITOR BCL-2 AND ITS HOMOLOGUES INFLUENCE CONTROL OF CELL CYCLE ENTRY

The effect of the cell death inhibitor Bcl‐2 and its homologues on cell cycle regulation was explored in lymphocytes and cell lines. Expression of a bcl‐2 transgene reduced proliferation of thymocytes and delayed cell cycle entry of mitogen‐stimulated B and T lymphocytes. Overexpression of Bcl‐2, Bcl‐xL or adenovirus E1B19kD substantially delayed serum stimulation‐induced S phase entry of quiescent NIH 3T3 fibroblasts. Bcl‐2‐mediated cell survival and growth inhibition are both antagonized by Bax. Bcl‐2, Bcl‐xL and E1B19kD, but not Bcl‐2 mutants that are defective in blocking apoptosis, suppress growth of colon carcinoma cells. This evidence that regulation of cell survival is coupled to control of cell growth has implications for normal cell turnover and tumorigenesis.

The anti-apoptosis function of Bcl-2 can be genetically separated from its inhibitory effect on cell cycle entry

The Bcl‐2 family of proteins regulate apoptosis, some antagonizing cell death and others facilitating it. It has recently been demonstrated that Bcl‐2 not only inhibits apoptosis but also restrains cell cycle entry. We show here that these two functions can be genetically dissociated. Mutation of a tyrosine residue within the conserved N‐terminal BH4 region had no effect on the ability of Bcl‐2 or its closest homologs to enhance cell survival and did not prevent heterodimerization with death‐enhancing family members Bax, Bak, Bad and Bik. Neither did this mutation override the growth‐inhibitory effect of p53. However, on stimulation with cytokine or serum, starved quiescent cells expressing the mutant proteins re‐entered the cell cycle much faster than those expressing comparable levels of wild‐type proteins. When wild‐type and Y28 mutant Bcl‐2 were co‐expressed, the mutant was dominant. Although R‐Ras p23 has been reported to bind to Bcl‐2, no interaction was detectable in transfected cells and R‐Ras p23 did not interfere with the ability of Bcl‐2 to inhibit apoptosis or cell cycle entry. These observations provide evidence that the anti‐apoptotic function of Bcl‐2 is mechanistically distinct from its inhibitory influence on cell cycle entry.

The anti‐apoptotic activities of Rel and RelA required during B‐cell maturation involve the regulation of Bcl‐2 expression

Rel and RelA, individually dispensable for lymphopoiesis, serve unique functions in activated B and T cells. Here their combined roles in lymphocyte development were examined in chimeric mice repopulated with c‐rel−/− rela−/− fetal liver hemopoietic stem cells. Mice engrafted with double‐mutant cells lacked mature IgMloIgDhi B cells, and numbers of peripheral CD4+ and CD8+ T cells were markedly reduced. The absence of mature B cells was associated with impaired survival that coincided with reduced expression of bcl‐2 and A1. bcl‐2 transgene expression not only prevented apoptosis and increased peripheral B‐cell numbers, but also induced further maturation to an IgMloIgDhi phenotype. In contrast, the survival of double‐mutant T cells was normal and the bcl‐2 transgene could not rectify the peripheral T‐cell deficit. These findings indicate that Rel and RelA serve essential, albeit redundant, functions during the later antigen‐independent stages of B‐ and T‐cell maturation, with these transcription factors promoting the survival of peripheral B cells in part by upregulating Bcl‐2.

Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function

Procoagulant platelets exhibit hallmark features of apoptotic cells, including membrane blebbing, microvesiculation, and phosphatidylserine (PS) exposure. Although platelets possess many well-known apoptotic regulators, their role in regulating the procoagulant function of platelets is unclear. To clarify this, we investigated the consequence of removing the essential mediators of apoptosis, Bak and Bax, or directly inducing apoptosis with the BH3 mimetic compound ABT-737. Treatment of platelets with ABT-737 triggered PS exposure and a marked increase in thrombin generation in vitro. This increase in procoagulant function was Bak/Bax- and caspase-dependent, but it was unaffected by inhibitors of platelet activation or by chelating extracellular calcium. In contrast, agonist-induced platelet procoagulant function was unchanged in Bak(-/-)Bax(-/-) or caspase inhibitor-treated platelets, but it was completely eliminated by extracellular calcium chelators or inhibitors of platelet activation. These studies show the existence of 2 distinct pathways regulating the procoagulant function of platelets.

Caspase-2 is not required for thymocyte or neuronal apoptosis even though cleavage of caspase-2 is dependent on both Apaf-1 and caspase-9

We have generated rat monoclonal antibodies that specifically recognise caspase-2 from many species, including mouse, rat and humans. Using these antibodies, we have investigated caspase-2 expression, subcellular localisation and processing. We demonstrate that caspase-2 is expressed in most tissues and cell types. Cell fractionation and immunohistochemistry experiments show that caspase-2 is found in the nuclear and cytosolic fractions, including a significant portion present in the Golgi complex. We found that caspase-2 is processed in response to many apoptotic stimuli but experiments with caspase-2 deficient mice demonstrated that it is not required for apoptosis of thymocytes or dorsal root ganglia (DRG) neurons in response to a variety of cytotoxic stimuli. Caspase-2 processing does not occur in thymocytes lacking Apaf-1 or caspase-9, suggesting that in this cell type, activation of caspase-2 occurs downstream of apoptosome formation.

The Dendritic Cell Receptor Clec9A Binds Damaged Cells via Exposed Actin Filaments

The immune system must distinguish viable cells from cells damaged by physical and infective processes. The damaged cell-recognition molecule Clec9A is expressed on the surface of the mouse and human dendritic cell subsets specialized for the uptake and processing of material from dead cells. Clec9A recognizes a conserved component within nucleated and nonnucleated cells, exposed when cell membranes are damaged. We have identified this Clec9A ligand as a filamentous form of actin in association with particular actin-binding domains of cytoskeletal proteins. We have determined the crystal structure of the human CLEC9A C-type lectin domain and propose a functional dimeric structure with conserved tryptophans in the ligand recognition site. Mutation of these residues ablated CLEC9A binding to damaged cells and to the isolated ligand complexes. We propose that Clec9A provides targeted recruitment of the adaptive immune system during infection and can also be utilized to enhance immune responses generated by vaccines.