Signal transduction in response to interleukin‐6 (IL‐6) requires binding of the cytokine to its receptor (IL‐6R) and subsequent homodimerization of the signal transducer gp130. The complex of IL‐6 and soluble IL‐6R (sIL‐6R) triggers dimerization of gp130 and induces responses on cells that do not express membrane bound IL‐6R. Naturally occurring soluble gp130 (sgp130) can be found in a ternary complex with IL‐6 and sIL‐6R. We created recombinant sgp130 proteins that showed binding to IL‐6 in complex with sIL‐6R and inhibited IL‐6/sIL‐6R induced proliferation of BAF/3 cells expressing gp130. Surprisingly, sgp130 proteins did not affect IL‐6 stimulated proliferation of BAF/3 cells expressing gp130 and membrane bound IL‐6R, indicating that sgp130 did not interfere with IL‐6 bound to IL‐6R on the cell surface. Additionally, sgp130 partially inhibited proliferation induced by leukemia inhibitory factor (LIF) and oncostatin M (OSM) albeit at higher concentrations. Recombinant sgp130 protein could be used to block the anti‐apoptotic effect of sIL‐6R on lamina propria cells from Crohn disease patients. We conclude that sgp130 is the natural inhibitor of IL‐6 responses dependent on sIL‐6R. Furthermore, recombinant sgp130 is expected to be a valuable therapeutic tool to specifically block disease states in which sIL‐6R transsignaling responses exist, e.g. in morbus Crohn disease.

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 has been shown for hematopoietic progenitor cells, endothelial cells, and smooth muscle cells. In this paper we show that purified recombinant vIL-6 binds to gp130 and stimulates primary human smooth muscle cells. IL-6R fails to bind vIL-6 and is not involved in its signaling. A Fc fusion protein of gp130 turned out to be a potent inhibitor of vIL-6. Our data demonstrate that vIL-6 is the first cytokine which directly binds and activates gp130. This property points to a possible role of this viral cytokine in the pathophysiology of human herpes virus 8.

Although regulated ectodomain shedding affects a large panel of structurally and functionally unrelated proteins, little is known about the mechanisms controlling this process. Despite a lack of sequence similarities around cleavage sites, most proteins are shed in response to the stimulation of protein kinase C by phorbol esters. The signal-transducing receptor subunit gp130 is not a substrate of the regulated shedding machinery. We generated several chimaeric proteins of gp130 and the proteins tumour necrosis factor alpha (TNF-alpha), transforming growth factor alpha (TGF-alpha) and interleukin 6 receptor (IL-6R), which are known to be subject to shedding. By exchanging small peptide sequences of gp130 for cleavage-site peptides of TNF-alpha, TGF-alpha and IL-6R we showed that these short sequences conferred susceptibility to spontaneous and phorbol-ester-induced shedding of gp130. Importantly, these chimaeric gp130 proteins were functional, as shown by the phosphorylation of gp130 and the activation of signal transduction and activators of transcription 3 ('STAT3') on stimulation with cytokine. To investigate minimal requirements for shedding, truncated cleavage-site peptides of IL-6R were inserted into gp130. The resulting chimaeras were susceptible to shedding and showed the same cleavage pattern as observed in the chimaeras containing the complete IL-6R cleavage site. Surprisingly, we could also generate cleavable chimaeras by exchanging the juxtamembrane sequence of gp130 for the corresponding region of leukaemia inhibitory factor ('LIF') receptor, a protein that like gp130 is not subject to regulated or spontaneous shedding. Thus it seems that there is no minimal consensus shedding sequence. We speculate that structural changes allow the access of the protease to a membrane-proximal region, leading to shedding of the protein.

A functionally and structurally diverse group of transmembrane proteins including transmembrane forms of mediators or receptors can be proteolytically cleaved to form soluble growth factors or receptors. Recently, the proteolytic activity responsible for pro-tumor necrosis factor a (proTNFa) processing has been identified and named TACE (TNFa converting enzyme). In experiments with TACE deficient (TACE -/-) fibroblasts we found that 4b-phorbol 12-myristate 13-acetate (PMA)-induced shedding of the interleukin-6 receptor (IL-6R) is strongly reduced. A basal hydroxamate sensitive release of IL-6R, however, could still be detected. This result demonstrates that TACE plays a role in IL-6R processing and that additional metalloproteases might be involved. PMA-induced shedding of IL-6R in TACE deficient mouse fibroblasts could be restored by stable transfection of a TACE cDNA. To characterize differences between shedding of IL-6R and proTNFa we generated chimeric IL-6R and proTNFa proteins wherein the endogenous cleavage sites (CS) had been replaced by the corresponding region of proTNFa and IL-6R, respectively. Interestingly, proTNFa chimeric proteins showed only minimal shedding. In contrast, IL-6R chimeras containing the proTNFa CS were shed spontaneously, processing was not further induced by PMA. Thus, the cleavage pattern transferred by the introduction of the proTNFa CS is similar to that of proTNFa itself. We conclude that the amino-acid sequence at the proteolytic CS contributes to the cleavage characteristics of a protein. However, this information alone is not sufficient to transfer cleavability as seen with proTNFa chimeras containing the IL-6R CS and which were resistant to 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 a receptor and the common glycoprotein (gp)130 subunit for signal transduction. The extracellular parts of the a-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-6RDIg and soluble IL-6RDIg). IL-6RDIg was shed as effectively as wild-type IL-6R from transfected COS-7 cells upon 4b-phorbol 12-myristate 13-acetate (PMA) treatment, whereas nonstimulated shedding of IL-6RDIg was not observed. The shed sIL-6RDIg from PMA-treated cells, as well as the transmembrane IL-6RDIg, had the same biological activity as wild-type sIL-6R, as measured by the induction of haptoglobin secretion in HepG2-IL-6 cells and IL-6-dependent proliferation of IL-6RDIg transfected BAF/gp130 cells. In COS-7 cells transfected with IL-6RDIg or soluble IL-6RDIg cDNA, transport of the deletion mutants through the secretory pathway appeared to be delayed because a sizeable proportion of the mutants was detected as an endo-b-N-acetylglucosaminidase-sensitive intermediate, suggesting that transport and processing of the DIg mutants on the secretory pathway were impaired. These experiments suggest that the Ig-like domain of the IL-6R is important for intracellular transport of IL-6R through the secretory pathway. Furthermore, the Ig-like domain is necessary for noninduced shedding of the IL-6R, whereas it has no function in PKC-dependent shedding of the IL-6R.