Wim Declercq

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.

Characterization of seven murine caspase family members

© 1997 Federation of European Biochemical Societies.

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.

Development of a simple, sensitive and specific bioassay for interleukin-1 based on the proliferation of RPMI 1788 cells comparison with other bioassays for IL-1

The IL-1-dependent proliferation of RPMI 1788, a human EBV-transformed cell line, was used to develop a biological assay system for IL-1. Preparations of rhIL-1 alpha and rhIL-1 beta, as well as rmIL-1 beta exhibited a specific biological activity (50% of the maximal response) between 5.8 x 10(8) and 8.6 x 10(8) U/mg. Remarkably, a 3-5-fold reduced specific biological activity was noticed for rm-IL-1 alpha, viz. 1.7 x 10(8) U/mg. The IL-1-dependent proliferation of RPMI 1788 cells was compared with other IL-1 test systems, such as the IL-1-mediated induction of IL-2 in EL4-NOB-1, LBRM-33-1A5 and thymocytes, and the IL-1-driven induction of cytotoxic activity by PC60 cells, the so-called CIA assay. The cytokine-dependent growth of RPMI 1788 cells is highly specific for IL-1, and no other cytokine tested induced a proliferative response. The presence of high concentrations of rmTNF, rhTNF or rhIL-6 did not interfere with the quantification of IL-1. Additionally, we evaluated the detection of IL-1 in the presence of mitogens, phorbol ester or calcium ionophore, as well as the determination of IL-1 in serum and PF samples of human and murine origin.