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

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https://journals-co-za.ez.sun.ac.za/doi/10.47348/SAMLJ/v33/i2a3Electronic forma

Der Interleukin-6 Rezeptor: Spezies-spezifische Unterschiede der proteolytischen Freisetzung durch die Metalloproteasen ADAM10 und ADAM17 zwischen Maus und Mensch

Interleukin-6 (IL-6) übernimmt sowohl bei physiologischen als auch bei pathophysiologischen Prozessen eine Schlüsselrolle in der Koordination von Immunantworten. Ein wichtiger Schritt ist hierbei das so genannte IL-6 Trans-signaling, bei dem IL-6 an den löslichen IL-6 Rezeptor (sIL-6R) bindet. Der entstandene Komplex ist in der Lage, praktisch alle Zellen durch Bindung an den membranständigen signaltransduzierenden Rezeptor 130 (gp130) zu aktivieren. IL-6 alleine kann dies nur bei Zellen, die den membrangebundenen IL-6R tragen. Löslicher IL-6R entsteht im Menschen hauptsächlich durch limitierte Proteolyse des membranständigen Rezeptors (Shedding), aber auch durch alternatives Splicen der IL-6R mRNA. Es ist bekannt, dass der humane IL-6R von ADAM17, einem Mitglied der A Disintegrin And Metalloprotease Proteinfamilie, und zu einem geringeren Teil von ADAM10 prozessiert wird. Man nimmt an, dass ADAM10 hierbei für das langsame, konstitutive Shedding verantwortlich ist, während ADAM17 den IL-6R schnell nach (patho-)physiologischer Stimulation schneidet. Analoge Daten zum murinen IL-6R gibt es bislang nicht. In dieser Arbeit wurde gezeigt, dass der IL-6R in der Maus nur in geringem Maße durch ADAM17 proteolytisch gespalten wird. Hypomorphe ADAM17ex/ex-Mäuse, die eine um rund 95 % reduzierte ADAM17 Expression in allen Geweben aufweisen, zeigten keine Reduktion des Serumspiegels an löslichem murinem IL-6R (smIL-6R) verglichen mit Wildtyp-Mäusen. Stimulation mit dem Phorbolester PMA war zum großen Teil nicht in der Lage, ADAM17-mediiertes Shedding des mIL-6R auszulösen, was unterstreicht, dass murines ADAM17 (mADAM17) nicht die Hauptprotease des mIL-6R ist. Shedding des mIL-6R durch ADAM17 konnte hingegen erzielt werden, wenn Teile des extrazellulären Bereiches durch ihr humanes Gegenstück ausgetauscht wurden. Die Transmembran- und die intrazelluläre Domäne hatten hierauf keinen Einfluss. Dieses deutet auf eine wichtige Rolle des extrazellulären Bereichs des IL-6R in Bezug auf die Erkennung des Substrats IL-6R durch seine Protease hin. Kürzlich wurde Apoptose als natürlicher Stimulus entdeckt, der ADAM17-mediiertes Shedding des hIL-6R verursacht und damit zur Auflösung einer akuten Entzündung beiträgt. Obwohl Apoptose ebenso das Shedding des IL-6R in der Maus auslöst, wurde die verantwortliche Protease im Rahmen dieser Arbeit als murines ADAM10 (mA- DAM10) identifiziert. ADAM10 war ebenso verantwortlich für das durch Ionomycin ausgelöste Shedding von humanem und murinem IL-6R. In murinen embryonalen Fibroblasten, die defizient für ADAM10 sind, wurde die Proteolyse des humanen IL-6R von ADAM17 kompensiert, wo hingegen muriner IL-6R von einer bisher noch nicht identifizierten weiteren Protease geschnitten wurde. In weiteren Experimenten konnte die physiologische Stimulation des purinergen P2X7-Rezeptors als neuer Stimulus der IL-6R Proteolyse identifiziert werden, die ausschließlich durch humanes und murines ADAM10 vermittelt wurde. Dieser neue Signalweg trug zum physiologischen Serumlevel an smIL-6R in der Maus bei, was durch signifikant reduzierte Werte an smIL-6R im Blut P2X7-R defizienter Mäuse gezeigt werden konnte. Schließlich wurde das stress- auslösende Antibiotikum Anisomycin als weiterer Stimulus der Proteolyse des hIL-6R durch ADAM17 mittels Aktivierung der p38 MAPK identifiziert. Für diesen Prozess war die intrazelluläre Domäne (ICD) des IL-6R erforderlich. Es stellt somit den ersten beschriebenen Prozess dar, in dem die ICD des IL-6R für das Shedding erforderlich ist. Zusammenfassend zeigt die vorliegende Arbeit eine unerwartete Speziesspezifität von ADAM10 und ADAM17 beim Shedding des IL-6R und identifiziert ADAM10 als neue induzierbare Protease des IL-6R in Mensch und Maus. Dies kann Konsequenzen für die Interpretation von Phenotypen von ADAM17 und ADAM10 defizienten Mäusen, insbesondere bei Krankheitsmodellen, haben.Interleukin-6 (IL-6) is an important mediator of immune responses both during physiologic and pathophysiologic processes. A key event is the so-called IL-6 trans-signaling, in which the soluble Interleukin-6 receptor (sIL-6R) binds to IL-6. This complex is able to activate virtually all cells through binding to the membrane-bound glycoprotein 130 (gp130), whereas IL-6 alone is only able to activate cells expressing the membrane-bound IL-6R. In humans, the sIL-6R is predominantly generated by proteolytic cleavage of the membrane-anchored form (shedding) and to a minor extent by alternative splicing. It is known from previous studies that shedding of the human IL-6R is mediated by A Disintegrin And Metalloprotease 17 (ADAM17) and to a minor extent by ADAM10. It is believed that hereby ADAM10 is responsible for the slow, constitutive shedding, whereas ADAM17 is activated through (patho-)physiological stimuli. Comparable data for murine IL-6R are missing. In this study, it was shown that ADAM17 is not the main sheddase of murine IL-6R in mice. In hypomorphic ADAM17ex/ex mice, which have about 95 % reduced ADAM17, serum levels of soluble murine IL-6R (smIL-6R) were not reduced compared to wildtype mice. Phorbol ester stimulation was largely ineffective to induce ADAM17-mediated shedding of mIL-6R, indicating that murine ADAM17 (mADAM17) was not the major sheddase of murine IL-6R. Shedding of mIL-6R by mADAM17 was rescued in chimeric mIL-6R proteins containing any extracellular domain of human IL-6R but not the transmembrane or the intracellular domain. This points to a role of the extracellular domains of IL-6R in substrate/protease recognition. Recently, apoptosis was identified as a physiological stimulus of ADAM17-mediated shedding of hIL-6R and IL-6 trans-signaling for the resolution of acute inflammation. Even though apoptosis induced mIL-6R shedding in mice, the responsible protease was identified as murine ADAM10 (mADAM10). ADAM10 was also identified as the protease responsible for Ionomycin-induced shedding of human and murine IL-6R. However, in ADAM10 deficient murine embryonic fibroblasts compensatory shedding of hIL-6R was mediated by ADAM17 but loss of ADAM10-mediated shedding of mIL-6R was compensated by a yet unidentified protease. Moreover, physiological stimulation of the purinergic P2X7 receptor was discovered as a novel inducer of IL-6R shedding solely mediated by murine and human ADAM10. This pathway contributed to the physiologic serum level of sIL-6R in mice as shown by significant reduction of smIL-6R in the blood of P2X7-R deficient mice. Finally, the stress-inducing antibiotic anisomycin was shown to induce shedding of hIL-6R via ADAM17 depending on the activation of p38 MAPK, a process, in which the intracellular domain (ICD) of the IL-6R was needed. This showed for the first time the requirement of the ICD of the human IL-6R for a shedding event. In conclusion, this work showed an unexpected species specificity of ADAM10 and ADAM17 and identified ADAM10 as novel inducible sheddase of IL-6R in mice and men, which might have consequences for the interpretation of phenotypes from ADAM17 and ADAM10 deficient mice, especially for animal models of human diseases

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

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

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

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

Identification of IL-6 Signalling Components as Predictors of Severity and Outcome in COVID-19

IL-6 is one of the major mediators of the hyper-inflammatory responses with complex biological functions as it can signal via different modes of action. IL-6 by classical signalling has anti-inflammatory and antibacterial activities, while trans-signalling mediates pro-inflammatory effects. The net biological effect of IL-6 is established by multiple factors beyond its absolute concentration. Here, we assess the relationship between IL-6 signalling variables [IL-6, soluble IL-6R (sIL-6R) and soluble gp130 (sgp130)] and outcomes in a cohort of 366 COVID-19 patients. The potential trans-signalling was evaluated by a ratio between the pro-inflammatory binary IL-6:sIL-6R complex and the inactive ternary IL-6:sIL-6R:sgp130 complex (binary/ternary complex) and the fold molar excess of sgp130 over sIL-6R (FME). Our data provide new evidence that high levels of IL-6, sIL-6R, sgp130, binary/ternary complex ratio, and low FME are independent predictors of COVID-19 severity in survivor patients (without death), and the combination of IL-6 + sIL-6R + sgp130 exhibited the most robust classification capacity. Conversely, in a subgroup of patients with a very poor prognosis, we found that high levels of IL-6 and low levels of sIL-6R, sgp130, and binary/ternary complex ratio were predictors of death. In this context, the highest predictive capacity corresponded to the combined analysis of IL-6 + FME + lymphopenia + creatinine. Herein, we present IL-6 signalling variables as a helpful tool for the early identification and stratification of patients with clear implications for treatment and clinical decision-making.The research work was supported by the Spanish Institute of Health Carlos III (COV-20/00792) and by the European Commission - NextgenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global). MAR-H acknowledges support from the Spanish Institute of Health Carlos III and the European Commission - NextgenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global). DC is supported by a predoctoral iPFIS (IFI 19/00048) funded by Spanish Institute of Health Carlos III. MN-N is supported by the Rio Hortega contract (CM20/00074)