Signal transduction of Interleukin-11 and Interleukin-6 ?-Receptors

The cytokines Interleukin (IL)-11 and IL-6 are important mediators that regulate differentiation and proliferation of immune cells. Both cytokines bind to unique non-signaling ?-receptors (IL-11R and IL-6R, respectively), and the resulting cytokine/cytokine receptor complexes recruit a homodimer of the signal-transducing ?-receptor glycoprotein (gp)130. Gp130 is expressed ubiquitously, whereas both ?-receptors show a cell- and tissue-specific expression pattern, thus determining cellular responsiveness towards IL-6 and/or IL-11. Formation of the signaling complexes activates intracellular signaling cascades, most prominently the Janus kinase (Jak)/Signal Transducer and Activator of Transcription (STAT) pathway. In a recent paper published in Biochimie, we analyzed the signaling capacity of eight chimeric receptors consisting of different domains of IL-11R and IL-6R. Our results showed that the intracellular region, the transmembrane region or the stalk region can be swapped between the two receptors, as they are not essential to discriminate between the two cytokines. Selectivity of the two receptors is exclusively warranted by the cytokine binding module (CBM), which resides within the domains D1 to D3. These results underline a modular organization of IL-11R and IL-6R and a comparable signal transduction of both cytokines

Interleukin-11 (IL-11) receptor cleavage by the rhomboid protease RHBDL2 induces IL-11 trans-signaling

Interleukin-11 (IL-11) is a pleiotropic cytokine with both pro- and anti-inflammatory properties. It activates its target cells via binding to the membrane-bound IL-11 receptor (IL-11R), which then recruits a homodimer of the ubiquitously expressed, signal-transducing receptor gp130. Besides this classic signaling pathway, IL-11 can also bind to soluble forms of the IL-11R (sIL-11R), and IL-11/sIL-11R complexes activate cells via the induction of gp130 homodimerization (trans-signaling). We have previously reported that the metalloprotease ADAM10 cleaves the membrane-bound IL-11R and thereby generates sIL-11R. In this study, we identify the rhomboid intramembrane protease RHBDL2 as a so far unrecognized alternative sheddase that can efficiently trigger IL-11R secretion. We determine the cleavage site used by RHBDL2, which is located in the extracellular part of the receptor in close proximity to the plasma membrane, between Ala-370 and Ser-371. Furthermore, we identify critical amino acid residues within the transmembrane helix that are required for IL-11R proteolysis. We also show that ectopically expressed RHBDL2 is able to cleave the IL-11R within the early secretory pathway and not only at the plasma membrane, indicating that its subcellular localization plays a central role in controlling its activity. Moreover, RHBDL2-derived sIL-11R is biologically active and able to perform IL-11 trans-signaling. Finally, we show that the human mutation IL-11R-A370V does not impede IL-11 classic signaling, but prevents RHBDL2-mediated IL-11R cleavage

Differences in Shedding of the Interleukin-11 Receptor by the Proteases ADAM9, ADAM10, ADAM17, Meprin α, Meprin β and MT1-MMP

Interleukin-11 (IL-11) has been associated with inflammatory conditions, bone homeostasis, hematopoiesis, and fertility. So far, these functions have been linked to classical IL-11 signaling via the membrane bound receptor (IL-11R). However, a signaling cascade via the soluble IL-11R (sIL-11R), generated by proteolytic cleavage, can also be induced. This process is called IL-11 trans-signaling. A disintegrin and metalloprotease 10 (ADAM10) and neutrophil elastase were described as ectodomain sheddases of the IL-11R, thereby inducing trans-signaling. Furthermore, previous studies employing approaches for the stimulation and inhibition of endogenous ADAM-proteases indicated that ADAM10, but not ADAM17, can cleave the IL-11R. Herein, we show that several metalloproteases, namely ADAM9, ADAM10, ADAM17, meprin β, and membrane-type 1 matrix metalloprotease/matrix metalloprotease-14 (MT1-MMP/MMP-14) when overexpressed are able to shed the IL-11R. All sIL-11R ectodomains were biologically active and capable of inducing signal transducer and activator of transcription 3 (STAT3) phosphorylation in target cells. The difference observed for ADAM10/17 specificity compared to previous studies can be explained by the different approaches used, such as stimulation of protease activity or making use of cells with genetically deleted enzymes

Characterization of the Cancer-Associated Meprin Beta Variants G45R and G89R

Meprin β is a metalloprotease associated with neurodegeneration, inflammation, extracellular matrix homeostasis, transendothelial cell migration, and cancer. In this study, we investigated two melanoma-associated variants of meprin β, both exhibiting a single amino acid exchange, namely, meprin β G45R and G89R. Based on the structural data of meprin β and with regard to the position of the amino acid exchanges, we hypothesized an increase in proteolytic activity in the case of the G45R variant due to the induction of a potential new activation site and a decrease in proteolytic activity from the G89R variant due to structural instability. Indeed, the G89R variant showed, overall, a reduced expression level compared to wild-type meprin β, accompanied by decreased activity and lower cell surface expression but strong accumulation in the endoplasmic reticulum. This was further supported by the analysis of the shedding of the interleukin-6 receptor (IL-6R) by meprin β and its variants. In transfected HEK cells, the G89R variant was found to generate less soluble IL-6R, whereas the expression of meprin β G45R resulted in increased shedding of the IL-6R compared to wild-type meprin β and the G89R variant. A similar tendency of the induced shedding capacity of G45R was seen for the well-described meprin β substrate CD99. Furthermore, employing an assay for cell migration in a collagen IV matrix, we observed that the transfection of wild-type meprin β and the G45R variant resulted in increased migration of HeLa cells, while the G89R variant led to diminished mobility

Kinetics of cytokine receptor trafficking determine signaling and functional selectivity

Cytokines activate signaling via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered IL-6 variants with different affinities to gp130 to investigate how cytokine receptor binding dwell-times influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes and induced biased signaling, with changes in receptor binding dwell-times affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This leads to a graded gene expression response and differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy

Natural Glycoforms of Human Interleukin 6 show atypical plasma clearance

A library of glycoforms of human interleukin 6 (IL‐6) comprising complex and mannosidic N‐glycans was generated by semisynthesis. The three segments were connected by sequential native chemical ligation followed by two‐step refolding. The central glycopeptide segments were assembled by pseudoproline‐assisted Lansbury aspartylation and subsequent enzymatic elongation of complex N‐glycans. Nine IL‐6 glycoforms were synthesized, seven of which were evaluated for in vivo plasma clearance in rats and compared to non‐glycosylated recombinant IL‐6 from E. coli. Each IL‐6 glycoform was tested in three animals and reproducibly showed individual serum clearances depending on the structure of the N‐glycan. The clearance rates were atypical, since the 2,6‐sialylated glycoforms of IL‐6 cleared faster than the corresponding asialo IL‐6 with terminal galactoses. Compared to non‐glycosylated IL‐6 the plasma clearance of IL‐6 glycoforms was delayed in the presence of larger and multibranched N‐glycans in most case

The interleukin-6 receptor Asp358Ala single nucleotide polymorphism rs2228145 confers increased proteolytic conversion rates by ADAM proteases

The pleiotropic activities of Interleukin (IL-)6 are controlled by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The coding single nucleotide polymorphism (SNP) rs2228145 of the Interleukin 6 receptor (IL-6R Asp358Ala variant) is associated with a 2-fold increase in soluble IL-6R (sIL-6R) serum levels resulting in reduced IL-6-induced C-reactive protein (CRP) production and a reduced risk for coronary heart disease. It was suggested that the increased sIL-6R level leads to decreased IL-6 classic or increased IL-6 trans-signaling. Irrespective of the functional outcome of increased sIL-6R serum level, it is still under debate, whether the increased sIL-6R serum levels emerged from differential splicing or ectodomain shedding. Here we show that increased proteolytic ectodomain shedding mediated by the A Disintegrin and metalloproteinase domain (ADAM) proteases ADAM10 and ADAM17 caused increased sIL-6R serum level in vitro as well as in healthy volunteers homozygous for the IL-6R Asp358Ala allele. Differential splicing of the IL-6R appears to have only a minor effect on sIL-6R level. Increased ectodomain shedding resulted in reduced cell-surface expression of the IL-6R Asp358Ala variant compared to the common IL-6R variant. In conclusion, increased IL-6R ectodomain shedding is a mechanistic explanation for the increased serum IL-6R levels found in persons homozygous for the rs2228145 IL-6R Asp358Ala variant

Generation of Soluble Interleukin-11 and Interleukin-6 Receptors: A Crucial Function for Proteases during Inflammation

The cytokines interleukin-11 (IL-11) and IL-6 are important proteins with well-defined pro- and anti-inflammatory functions. They activate intracellular signaling cascades through a homodimer of the ubiquitously expressed signal-transducing β-receptor glycoprotein 130 (gp130). Specificity is gained through the cell- and tissue-specific expression of the nonsignaling IL-11 and IL-6 α-receptors (IL-11R and IL-6R), which determine the responsiveness of the cell to these two cytokines. IL-6 is a rare example, where its soluble receptor (sIL-6R) has agonistic properties, so that the IL-6/sIL-6R complex is able to activate cells that are usually not responsive to IL-6 alone (trans-signaling). Recent evidence suggests that IL-11 can signal via a similar trans-signaling mechanism. In this review, we highlight similarities and differences in the functions of IL-11 and IL-6. We summarize current knowledge about the generation of the sIL-6R and sIL-11R by different proteases and discuss possible roles during inflammatory processes. Finally, we focus on the selective and/or combined inhibition of IL-6 and IL-11 signaling and how this might translate into the clinics