Rudi Beyaert

Two tumour necrosis factor receptors: structure and function

Tumour necrosis factor (TNF) exerts two main effects: a beneficial one as an anti-infection, anti-tumour cytokine, and a detrimental one in the systemic inflammatory response syndrome (SIRS). Two receptors (TNF-R) mediate these effects, but their precise role in different cell types is far from solved. TNF induces receptor oligomerization, an event that is believed to connect the receptors to downstream signalling pathways. Recent research suggests that several TNF-R-associated proteins, including kinases, may initiate cytoplasmic signal transduction.

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.

Molecular mechanisms of tumor necrosis factor-induced cytotoxicity - What we do understand and what we do not.

Although TNF plays an important role in several physiological and pathological conditions, the hallmark of this important cytokine has been its selective cytotoxic activity on tumor cells. Since its cloning in 1984, understanding of how TNF selectively kills tumor cells has been the subject of research in many laboratories. Here we review TNF‐induced post‐receptor signaling mechanisms which seem to be involved in the pathway to cytotoxicity.

Characterization of seven murine caspase family members

© 1997 Federation of European Biochemical Societies.

Reduced tumour necrosis factor-induced cytotoxicity by inhibitors of the arachidonic acid metabolism

The mechanism of tumour necrosis factor-mediated cytotoxicity was investigated by using various inhibitors of arachidonic acid metabolism. Phospholipase A2 inhibitors with different modes of action interfered with the cytotoxic action of TNF, whereas phospholipase C inhibitors did not. Neither cyclooxygenase nor lipoxygenase-blockers had a significant effect on TNF action. Experiments with scavengers of toxic oxygen radicals gave ambiguous results. The data obtained suggest the involvement of phospholipase A2 and arachidonic acid in the cytotoxic mechanism of TNF, but the exact role of these molecules is, however, still to be determined.

Casein kinase-1 phosphorylates the p75 tumor necrosis factor receptor and negatively regulates tumor necrosis factor signaling for apoptosis.

Cellular responses initiated by tumor necrosis factor (TNF) are mediated by two different cell surface receptors with respective molecular masses of 55 kDa (p55) and 75 kDa (p75). p55 is functional in almost every cell type and can independently transmit most biological activities of TNF. In contrast, TNF signaling via p75 seems so far largely restricted to cells of lymphoid origin, where it can induce proliferation, cytokine production, and/or apoptosis. The mechanisms that regulate TNF receptor activity are largely unknown. Here we report that the p75 of unstimulated p75-responsive PC60 T cells is phosphorylated on serine by a kinase activity present in p75 immune complexes. Several lines of evidence indicate that the latter kinase is casein kinase-1 (CK-1). Previous results have shown that the p75 TNF receptor is constitutively phosphorylated in vivo. Our data show that the latter in vivo phosphorylation is also at least partially due to CK-1. Pretreatment of cells with TNF had no detectable effect on p75 phosphorylation in vitro or in vivo. However, a specific CK-1 inhibitor potentiated TNF-induced apoptosis mediated by p75, suggesting an inhibitory role for phosphorylation by CK-1. Although in vivo p75 phosphorylation could be seen in both p75-unresponsive and p75-responsive cell lines, in vitro p75 phosphorylation in p75 coimmunoprecipitates could not be observed in cell lines that were biologically unresponsive to p75 stimulation. The latter observation further indicates a regulatory role for p75 phosphorylation in p75-mediated signaling. Taken together, our data demonstrate that the p75 TNF receptor is phosphorylated and associated with CK-1, which negatively regulates p75-mediated TNF signaling.

A20, an inhibitor of cell death, self-associates by its zinc finger domain.

A20 is a primary response gene which is induced after monocyte adherence or cytokine stimulation of a variety of cells. The A20 protein belongs to a novel class of Cys2/Cys2 zinc finger proteins, and has been characterized as an inhibitor of both apoptotic and necrotic cell death. In order to clarify its molecular mechanism of action, we used the yeast‐based‐two‐hybrid system to screen for A20‐associated proteins. Here we report that A20 is able to self‐associate, and demonstrate that the latter interaction is mediated by its zinc finger domain.

FUNCTIONAL CHARACTERIZATION OF THE PRODOMAIN OF INTERLEUKIN-1BETA -CONVERTING ENZYME

Interleukin-1β-converting enzyme (ICE) has been identified as the main protease responsible for maturation of the prodomain of interleukin-1β. Recently, it was shown to belong to a larger gene family, members of which play an important role in programmed cell death. A common feature of the ICE family proteases is the presence of a prodomain that has been hypothesized to keep the enzyme in an inactive form. Expression analysis in yeast revealed autocatalytic degradation of p45ICE, but not of p30ICE lacking a prodomain. We further demonstrate that p45ICE, in which the critical cysteine has been mutated, is still able to dimerize in vivo. Dimerization requires the prodomain and occurs prior to autoprocessing. These results provide evidence for a regulatory role of the prodomain of ICE.

Differential involvement of caspases in apoptosis of myeloid leukemic cells induced by chemotherapy versus growth factor withdrawal

To assess the potential involvement of the caspase family in the IL‐3‐dependent murine myeloid leukemic cell line 32D, we studied the effect of bcl‐2, crmA and three synthetic caspase inhibitors on apoptosis induced by chemotherapy or IL‐3 withdrawal. Apoptosis induced by IL‐3 deprivation or by ActD appears to be mediated by a crmA‐insensitive pathway. Cell death by IL‐3 withdrawal is inhibited by the caspase‐inhibitor ZVAD‐fmk, but not DEVD‐fmk or YVAD‐cmk. In contrast, DEVD‐fmk as well as ZVAD‐fmk protect 32D cells from ActD‐induced apoptosis. These results indicate that different caspases are involved in apoptosis induced by growth factor withdrawal and by chemotherapy.

Inhibition by glucocorticoids of tumor necrosis factor-mediated cytotoxicity: Evidence against lipocortin involvement

The role of the phospholipase inhibitor proteins, lipocortin‐I and ‐II, in tumor necrosis factor (TNF)‐mediated cytotoxicity against L929 fibrosarcoma cells was investigated. We previously reported that TNF‐mediated cytotoxicity was inhibited by dexamethasone (DEX), suggesting an involvement of lipocortins [1]. Now we show that, despite inhibition by DEX of TNF‐induced arachidonic acid release, DEX has no effect on the synthesis of these lipocortins. Moreover, TNF itself has no effect on the synthesis and phosphorylation of lipocortin‐I and ‐II. Also there was no difference in expression levels of lipocortin‐I and ‐II between TNF‐sensitive and ‐resistant cells. These data strongly suggest that the protective effect of DEX and other glucocorticoids is not mediated by lipocortins.The role of the phospholipase inhibitor proteins, lipocortin-I and -II, in tumor necrosis factor (TNF)-mediated cytotoxicity against L929 fibrosarcoma cells was investigated. We previously reported that TNF-mediated cytotoxicity was inhibited by dexamethasone (DEX), suggesting an involvement of lipocortins [1]. Now we show that, despite inhibition by DEX of TNF-induced arachidonic acid release, DEX has no effect on the synthesis of these lipocortins. Moreover, TNF itself has no effect on the synthesis and phosphorylation of lipocortin-I and -II. Also there was no difference in expression levels of lipocortin-I and -II between TNF-sensitive and -resistant cells. These data strongly suggest that the protective effect of DEX and other glucocorticoids is not mediated by lipocortins.