Cleavage of CD95 by matrix metalloproteinase-7 induces apoptosis resistance in tumour cells

The ability of tumour cells to resist apoptosis-inducing signals by cytotoxic T cells may decide the success or failure of tumour elimination. An important effector of apoptosis is the CD95/CD95 ligand system (APO-1/Fas) that mediates perforin-independent cytotoxic T-cell killing of tumour cells. We propose a new strategy by which tumour cells can resist CD95-induced apoptosis. We identified matrix metalloproteinase-7, MMP-7 (Martilysin), as the first physiologically relevant protease that can specifically cleave CD95. MMP-7 is of unique importance because it is produced by the tumour cells themselves at early stages of tumour development. Microsequencing of the positions in CD95 cleaved by…

Yeast expression of the cytokine receptor domain of the soluble interleukin-6 receptor

Abstract The complex of the soluble interleukin-6 receptor (sIL-6R) and IL-6 (IL-6) is a potent agonist on cells expressing the signal transducing protein gp130. In contrast, IL-6 alone only stimulates cells which express a membrane bound form of the IL-6R and gp130. The natural occurring sIL-6R is generated by shedding of the membrane receptor and to a lesser extend by alternative splicing. We have inserted the coding sequence of the 323 amino acid residues of the human sIL-6R into an expression/secretion vector suitable for the methylotrophic yeast Pichia pastoris . We obtained, however, no detectable expression and secretion of the recombinant protein. When we used only the coding sequen…

Human herpes virus 8 interleukin-6 homologue triggers gp130 on neuronal and hematopoietic cells

Human herpes virus-8 (HHV8) encodes a cytokine named viral interleukin-6 (vIL-6) that shares 25% amino-acid identity with its human homologue. Human IL-6 is known to be a growth and differentiation factor of lymphatic cells and plays a potential role in the pathophysiology of various lymphoproliferative diseases. vIL-6 is expressed in HHV8-associated-diseases including Kaposi's sarcoma, Body-cavity-based-lymphoma and Castleman's disease, suggesting a pathogenetic involvement in the malignant growth of B-cell associated diseases and other malignant tumours. We expressed vIL-6 in Escherichia coli as a fusion protein with recombinant periplasmic maltose binding protein. After cleavage from the…

The function of the soluble interleukin 6 (IL-6) receptor in vivo: sensitization of human soluble IL-6 receptor transgenic mice towards IL-6 and prolongation of the plasma half-life of IL-6.

Interleukin 6 (IL-6) is considered an important mediator of acute inflammatory responses. Moreover, IL-6 functions as a differentiation and growth factor of hematopoietic precursor cells, B cells, T cells, keratinocytes, neuronal cells, osteoclasts, and endothelial cells. IL-6 exhibits its action via a receptor complex consisting of a specific IL-6 receptor (IL-6R) and a signal transducing subunit (gp130). Soluble forms of both receptor components are generated by shedding and are found in patients with various diseases such as acquired immune deficiency syndrome, rheumatoid arthritis, and others. The function of the soluble (s)IL-6R in vivo is unknown. Since human (h)IL-6 acts on human and…

Generation and function of the soluble interleukin-6 receptor

Receptor Recognition Sites of Cytokines Are Organized as Exchangeable Modules

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-bindin…

Novel pathogenic mechanism of microbial metalloproteinases: liberation of membrane-anchored molecules in biologically active form exemplified by studies with the human interleukin-6 receptor

Certain membrane-anchored proteins, including several cytokines and cytokine receptors, can be released into cell supernatants through the action of endogenous membrane-bound metalloproteinases. The shed molecules are then able to fulfill various biological functions; for example, soluble interleukin-6 receptor (sIL-6R) can bind to bystander cells, rendering these cells sensitive to the action of IL-6. Using IL-6R as a model substrate, we report that the metalloproteinase from Serratia marcescens mimics the action of the endogenous shedding proteinase. Treatment of human monocytes with the bacterial protease led to a rapid release of sIL-6R into the supernatant. This effect was inhibitable …

Diverse cell surface protein ectodomains are shed by a system sensitive to metalloprotease inhibitors.

The extracellular domains of a diverse group of membrane proteins are shed in response to protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA). The lack of sequence similarity in the cleavage sites suggests the involvement of many proteases of diverse specificity in this process. However, a mutant Chinese hamster ovary cell line recently isolated for being defective in PMA-activated shedding of the membrane-anchored growth factor transforming growth factor alpha precursor (proTGF-alpha) is concomitantly defective in the shedding of many other unrelated membrane proteins. Here we show that independent mutagenesis and selection experiments yield shedding mutants having th…

IL-6 receptor independent stimulation of human gp130 by viral IL-6.

Abstract The genome of human herpes virus 8, which is associated with Kaposi’s sarcoma, encodes proteins with similarities to cytokines and chemokines including a homologue of IL-6. Although the function of these viral proteins is unclear, they might have the potential to modulate the immune system. For viral IL-6 (vIL-6), it has been demonstrated that it stimulates IL-6-dependent cells, indicating that the IL-6R system is used. IL-6 binds to IL-6R, and the IL-6/IL-6R complex associates with gp130 which dimerizes and initiates intracellular signaling. Cells that only express gp130 but no IL-6R cannot be stimulated by IL-6 unless a soluble form of the IL-6R is present. This type of signaling…

Pore-forming toxins trigger shedding of receptors for interleukin 6 and lipopolysaccharide.

Cleavage of membrane-associated proteins with the release of biologically active macromolecules is an emerging theme in biology. However, little is known about the nature and regulation of the involved proteases or about the physiological inducers of the shedding process. We here report that rapid and massive shedding of the interleukin 6 receptor (IL-6R) and the lipopolysaccharide receptor (CD14) occurs from primary and transfected cells attacked by two prototypes of pore-forming bacterial toxins, streptolysin O and Escherichia coli hemolysin. Shedding is not induced by an streptolysin O toxin mutant which retains cell binding capacity but lacks pore-forming activity. The toxin-dependent c…

A role for the immunoglobulin-like domain of the human IL-6 receptor. Intracellular protein transport and shedding.

Interleukin (IL)-6, IL-11 and cililary neurotrophic factor (CNTF) belong to the same family of hematopoietic and neurotrophic cytokines. Their receptor complexes contain a cytokine-binding alpha receptor and the common glycoprotein (gp)130 subunit for signal transduction. The extracellular parts of the alpha-receptor subunits consist of a membrane-proximal cytokine-binding domain and an N-terminal immunoglobulin (Ig)-like domain with unknown function. We examined the role of the Ig-like domain of IL-6R by constructing deletion mutants lacking the Ig domain (IL-6RDeltaIg and soluble IL-6RDeltaIg). IL-6RDeltaIg was shed as effectively as wild-type IL-6R from transfected COS-7 cells upon 4beta…

SHEDDING OF THE INTERLEUKIN-6 RECEPTOR: MECHANISMS AND PHYSIOLOGICAL CONSEQUENCES