Cell death induction by receptors of the TNF family: towards a molecular understanding

It has been known for a long time that programmed cell death (PCD) plays an essential role in the formation of a multicellular, differentiated animal 1. A striking example is the development of the nematode Caenorhabditis elegans. The fully formed animal consists of 1089 cells, of which 131 are programmed to die. This system has provided a breakthrough in understanding of the molecular processes underlying PCD. Indeed, genetic studies first revealed the presence of genes needed for PCD, such as ced-3 and ced-4, as well as other genes, such as ced-9, which counteract PCD 2. In addition to its role in development, PCD occurs prominently in various immune processes, such as negative selection of T-cells in the thymus, elimination of overactivated lymphocytes in the periphery, etc. But PCD—or lack of it—has especially become a focus of considerable interest because of its relevance to many diseases 3. For example, cancer cells have often become resistant to PCD-inducing stimuli, various lympho-proliferative and/or autoimmune diseases evade ‘death’ signals, etc. On the other hand, in some neuro-degenerative disorders such as Parkinson, Alzheimer or amyotrophic lateral sclerosis, one may wish to interfere with PCD. Cells dying in the course of development are in fact fairly difficult to study, because the cell corpses are rapidly phagocytosed by neighbouring cells. Therefore, morphological and biochemical processes associated with PCD can best be studied in cell culture systems, and the typical events leading to cell death are usually referred to as ‘apoptosis’. ‘PCD’ and ‘apoptosis’ are often used interchangeably, although the former has more a connotation of genetically predetermined, while the latter is more linked to morphological and biochemical changes 4. The principal characteristics of apoptosis are blebbing of the plasma membrane, phosphatidylserine externalization, cytoskeletal disruption, accumulation and/or activation of transglutaminase, condensation of nuclear chromatin, fragmentation of nuclear DNA to approximately 50 kb segments, and subsequently to internucleosomal fragments; at later stages, cytoplasm and nucleus become compartmentalized and form membrane-bound apoptotic bodies, which are engulfed by neighbouring cells or infiltrated tissue macrophages 4. Cell death by apoptosis is a very neat way to eliminate unwanted cells: no traces are left and the cell contents are never released or accessible to the immune system. Hence, there is no inflammation. This is in contrast to death by necrosis. Under these conditions, normally the cell swells and then, when membrane integrity comes under attack, the cell collapses like a balloon and the contents spill out into the extracellular milieu 5. This may result in an inflammatory response. Although PCD is sometimes referred to as ‘cell suicide’, in most systems studied the process of apoptosis is initiated by external inducers or treatments. Some examples are UV- or γ-irradiation, heat shock, oxidative stress, chemotherapeutic drugs (directed to topoisomerases like etoposide, to protein kinases like staurosporine, to DNA or its building blocks like nitrogen mustard and methotrexate, respectively, etc.), viral infection, loss of matrix attachment, glucocorticoids, growth factor withdrawal, soluble or membrane-bound cytokines like TNF or Fas/Apo-1 (CD95) ligand, and many others. In general, each cell contains precursors to apoptogenic mediators, as well as counteracting, protecting proteins. It is the balance between these two which determines the outcome after a given stimulus. It is remarkable how morphologically and biochemically similar apoptotically dying cells are, almost irrespective not only of the cell type, but also of the species. This suggests already that the molecular mechanisms of apoptosis are ubiquitous in nature and highly conserved in evolution. Indeed, various lines of evidence point to a universal, biochemical pathway. For example, the human anti-apoptotic protein Bcl-2 (to be discussed in a later section) can partially restore a defective ced-9 function in C. elegans 6, 7. Equally important, in most systems of apoptosis experimentally studied, clear evidence for involvement of caspases was found. Caspases are a recently discovered, extended family of Cysteine-type proteases, which cleave after an aspartic acid residue; the C. elegans CED-3 protein and interleukin-1β-converting enzyme (ICE) are typical examples. Strong evidence for the involvement of caspases is the fact that usually the process of apoptosis can be interfered with by specific caspase inhibitors, such as zVAD-fmk or viral antagonistic pseudo-substrates (see below). TNF and Fas/Apo-1 ligand play a prominent role in a variety of immunological, inflammatory and pathological conditions. Therefore, many studies on PCD have been done with TNF-R55 (CD120a) and Fas/Apo-1-induced death. Both receptors contain near their intracellular C-terminus a homologous region of about 90 amino acids, referred to as the ‘death domain’ (DD). Upon clustering, this DD is sufficient for signalling cell death. TNF-R55-DD in addition leads to activation of the transcription factor NF-κB and to gene induction. Many of the induced genes are important for inflammation (IL-6, IL-8, ICAM-1, E-selectin), while activation of NF-κB is also involved in synthesis of resistance proteins, such as A20 8. Recently, other receptors of the same family have been cloned and shown to contain a DD-related sequence, such as DR3/WSL-1/TRAMP 9-11, chicken CAR-1 12and DR4 13. The TNF receptor and ligand families are currently the object of intense research, covering a variety of different aspects of cellular signalling (reviewed in 14-20). These ligand and receptor families, although large and continuing to increase in size, display rather homogeneous of the involved in of the receptor family their a a in different in their extracellular but one of the growth factor their receptors a of in a These of are in similar mechanisms of and of the TNF ligand family and in each ligand to and Indeed, family are as a of of receptor and can be by their with of the TNF ligand family is that not only as soluble but also in with the of their cells. of these are as cell which the soluble are The only although formed as a soluble can as a to of the and also as a In contrast to the rather homogeneous of amino acid and protein involved in ligand receptor the amino acid of the intracellular of the TNF receptor family This to the that the receptors and mechanisms of the molecular understanding of the signalling by these receptors was that this was It was for example, that some of between the intracellular to be of which is not in a of receptors with intracellular have out to be of the same signalling In there to be rather intense between the different receptors of different signalling that to These are in a of function was This also to the of the receptors of the family to about this is to the function of which to TNF and which to the of Fas/Apo-1 evidence that such can also be by other receptors of the These the two other TNF receptors signalling plays an important role in the of lymphocytes that of and the receptor the formed between and and also DR3/WSL-1/TRAMP receptor to 9-11, the receptor and others. 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Although in of these cleave of for caspases are as precursors or The consists of a a more in the of and by a of approximately often a and a of approximately The is for of the and/or leads to the formation of evidence to a role of caspases in a variety of different apoptotic processes (reviewed in is a to that for a cell death process to in a programmed caspase involvement is a lines of evidence the involvement of caspases in cell death by TNF and cell death can be by of It can also be by two that as caspase inhibitors, the and the protein Caspases are as in other PCD processes, of cell death by TNF and Fas/Apo-1 has been to result in activation of caspases their that as caspase and have been shown to be in other apoptotic processes also to be at of the death processes induced by TNF or Fas/Apo-1 to the rather evidence that caspases play an important role in processes of death about the nature of this role is is on the mechanisms of activation of caspases in the of the cell death of caspase function in which is is the of the by Many of these are involved in the events leading to apoptotic cell death (to be discussed in of caspase of which have only understanding is the of their in cells Although there are also clear of between the their into These are in the of the evidence that the caspase activation and that their activation of different caspases by different The most evidence for this comes studies of the way in which caspases are by TNF-R55 and to the by which TNF-R55 and Fas/Apo-1 caspase have to the of caspases can to with these The of to in a ‘death domain’ or the of the DD in and to with of the in the proteins. 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