Interleukin-6 controls recycling and degradation, but not internalization of its receptors.

Interleukin-6 (IL-6) is a cytokine implicated in proinflammatory as well as regenerative processes and acts via receptor complexes consisting of the ubiquitously expressed, signal-transducing receptor gp130 and the IL-6 receptor (IL-6R). The IL-6R is expressed only on hepatocytes and subsets of leukocytes, where it mediates specificity of the receptor complex to IL-6 as the subunit gp130 is shared with all other members of the IL-6 cytokine family such as IL-11 or IL-27. The amount of IL-6R at the cell surface thus determines the responsiveness of the cell to the cytokine and might therefore be decisive in the development of inflammatory disorders. However, how the expression levels of IL-6R and gp130 at the cell surface are controlled is largely unknown. Here, we show that IL-6R and gp130 are constitutively internalized independent of IL-6. This process depends on dynamin and clathrin and is temporally controlled by motifs within the intracellular region of gp130 and IL-6R. IL-6 binding and internalization of the receptors is a prerequisite for activation of the Jak/STAT signaling cascade. Targeting of gp130, but not of the IL-6R, to the lysosome for degradation depends on stimulation with IL-6. Furthermore, we show that after internalization and activation of signaling, both the IL-6R and gp130 are recycled back to the cell surface, a process that is enhanced by IL-6. These data reveal an important function of IL-6 beyond the pure activation of signaling.

Proteolytic Origin of the Soluble Human IL-6R In Vivo and a Decisive Role of N-Glycosylation.

Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography-mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation.

The IL-6-neutralizing sIL-6R-sgp130 buffer system is disturbed in patients with type 2 diabetes.

Serum levels of interleukin-6 (IL-6) are increased in patients with type 2 diabetes (T2D). IL-6 exerts its pleiotropic effects via the IL-6 α-receptor (IL-6R), which exists in membrane-bound and soluble (sIL-6R) forms and activates cells via the ß-receptor glycoprotein 130 (gp130). The nonsynonymous single-nucleotide polymorphism (SNP) rs2228145 (Asp358Ala) within the IL6R locus is associated with T2D. The aim of this study was to determine whether sIL-6R in combination with soluble gp130 (sgp130) is able to form an IL-6-neutralizing buffer in healthy subjects and whether this is disturbed in T2D. We found that sIL-6R-sgp130 indeed forms an IL-6-neutralizing buffer in the serum of healthy humans, whose capacity is controlled by the SNP of the IL-6R. Circulating sIL-6R-sgp130 levels were lower in T2D subjects (P < 0.001), whereas IL-6 was high and inversely correlated with sIL-6R (r = -0.57, P < 0.001), indicating a severe disturbance of the buffer. This phenomenon is also observed in sex- and age-matched patients with both T2D and atherosclerosis but not in patients with atherosclerosis alone. In conclusion, sIL-6R and sgp130 serum levels were significantly lower in T2D patients compared with healthy subjects or atherosclerosis patients, although IL-6 levels were high. These data suggest that disturbance of the protective buffer may be closely associated with T2D pathophysiology.

Proteolytic control of Interleukin-11 and Interleukin-6 biology.

Interleukin-11 (IL-11) and IL-6 are secreted glycoproteins which fulfill important homeostatic functions. Activation of target cells occurs via membrane-bound IL-11 and IL-6 receptors (IL-11R and IL-6R, respectively). Formation of IL-11/IL-11R and IL-6/IL-6R complexes triggers the recruitment of a homodimer of the ubiquitously expressed signal-transducing ß-receptor gp130 (classic signaling). IL-11R and IL-6R can be shed by several proteases, albeit with different preferences and specificities, and these soluble receptors (sIL-11R and sIL-6R) act as agonists and can activate in principle all cells via gp130. We have termed these protease-controlled pathways IL-6 and IL-11 trans-signaling. In this review, we describe the basic biology of both cytokines and summarize the current knowledge how proteases control and shape the trans-signaling pathways of the two cytokines. We will further highlight how the underlying molecular mechanisms can be used to design specific inhibitors that block trans, but not classic signaling of IL-11 and IL-6. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Cleavage of the Interleukin-11 receptor induces processing of its C-terminal fragments by the gamma-secretase and the proteasome.

The cytokine Interleukin-11 (IL-11) signals through the membrane-bound IL-11 receptor (IL-11R), which is expressed in a cell-type specific manner. We have recently shown that the metalloprotease ADAM10 can cleave the IL-11R. The liberated soluble IL-11R (sIL-11R) ectodomain can bind its ligand, and the resulting IL-11/sIL-11R complex can activate cells that do not express the IL-11R (trans-signaling). In this study, we show that the remaining C-terminal fragment (CTF1) after ADAM10-mediated cleavage is subsequently cleaved within the membrane by the gamma-secretase complex, and that the resulting shorter CTF2 is further degraded by the proteasome. In contrast to other transmembrane receptors, e.g. Notch, we find no evidence that the IL-11R CTF can translocate into the nucleus to act as a transcription factor, suggesting that regulated intramembrane proteolysis of the IL-11R CTF acts as a mechanism to clear the plasma membrane from remaining protein fragments after cleavage of its ectodomain.

Cathepsin S provokes interleukin-6 (IL-6) trans-signaling through cleavage of the IL-6 receptor in vitro.

The cytokine interleukin-6 (IL-6) fulfills its pleiotropic functions via different modes of signaling. Regenerative and anti-inflammatory activities are mediated via classic signaling, in which IL-6 binds to the membrane-bound IL-6 receptor (IL-6R). For IL-6 trans-signaling, which accounts for the pro-inflammatory properties of the cytokine, IL-6 activates its target cells via soluble forms of the IL-6R (sIL-6R). We have previously shown that the majority of sIL-6R in human serum originates from proteolytic cleavage and mapped the cleavage site of the IL-6R. The cleavage occurs between Pro-355 and Val-356, which is the same cleavage site that the metalloprotease ADAM17 uses in vitro. However, sIL-6R serum levels are unchanged in hypomorphic ADAM17ex/ex mice, making the involvement of ADAM17 questionable. In order to identify other proteases that could be relevant for sIL-6R generation in vivo, we perform a screening approach based on the known cleavage site. We identify several candidate proteases and characterize the cysteine protease cathepsin S (CTSS) in detail. We show that CTSS is able to cleave the IL-6R in vitro and that the released sIL-6R is biologically active and can induce IL-6 trans-signaling. However, CTSS does not use the Pro-355/Val-356 cleavage site, and sIL-6R serum levels are not altered in Ctss-/- mice. In conclusion, we identify a novel protease of the IL-6R that can induce IL-6 trans-signaling, but does not contribute to steady-state sIL-6R serum levels.

Activation of Toll-like Receptor 2 (TLR2) induces Interleukin-6 trans-signaling.

Signaling of the pleiotropic cytokine Interleukin-6 (IL-6) via its soluble IL-6R (sIL-6R) has been termed trans-signaling and is thought to be responsible for the pro-inflammatory properties of IL-6. The sIL-6R can be generated by alternative mRNA splicing or proteolytic cleavage of the membrane-bound IL-6R. However, which stimuli induce sIL-6R release and which endogenous signaling pathways are required for this process is poorly understood. Here, we show that activation of Toll-like receptor 2 (TLR2) on primary human peripheral blood mononuclear cells (PBMCs) and on the monocytic cell line THP-1 induces expression and secretion of IL-6 and the generation of sIL-6R. We show by flow cytometry that monocytes are a PBMC subset that expresses TLR2 in conjunction with the IL-6R and are the major cellular source for both IL-6 and sIL-6R. Mechanistically, we find that the metalloproteases ADAM10 and ADAM17 are responsible for cleavage of the IL-6R and therefore sIL-6R generation. Finally, we identify the Extracellular-signal Regulated Kinase (ERK) cascade as a critical pathway that differentially regulates both IL-6 and sIL-6R generation in monocytes.

Mutations in Craniosynostosis Patients Cause Defective Interleukin-11 Receptor Maturation and Drive Craniosynostosis-like Disease in Mice.

Premature closure of the sutures that connect the cranial bones during development of the mammalian skull results in a phenotype called craniosynostosis. Recently, several craniosynostosis patients with missense mutations within the gene encoding the interleukin-11 receptor (IL-11R) have been described, but the underlying molecular mechanisms have remained elusive. IL-11 is a cytokine that has a crucial role in bone remodeling and activates cells via binding to the IL-11R. Here, we show that patient mutations prevented maturation of the IL-11R, resulting in endoplasmic reticulum retention and diminished cell surface appearance. Disruption of a conserved tryptophan-arginine zipper within the third domain of the IL-11R was the underlying cause of the defective maturation. IL-11 classic signaling via the membrane-bound receptor, but not IL-11 trans-signaling via the soluble receptor, was the crucial pathway for normal skull development in mice in vivo. Thus, the specific therapeutic inhibition of IL-11 trans-signaling does not interfere with skull development.

Control of ADAM17 activity by regulation of its cellular localisation.

An important, irreversible step in many signalling pathways is the shedding of membrane-anchored proteins. A Disintegrin And Metalloproteinase (ADAM) 17 is one of the major sheddases involved in a variety of physiological and pathophysiological processes including regeneration, differentiation, and cancer progression. This central role in signalling implies that ADAM17 activity has to be tightly regulated, including at the level of localisation. Most mature ADAM17 is localised intracellularly, with only a small amount at the cell surface. We found that ADAM17 is constitutively internalised by clathrin-coated pits and that physiological stimulators such as GPCR ligands induce ADAM17-mediated shedding, but do not alter the cell-surface abundance of the protease. In contrast, the PKC-activating phorbol ester PMA, often used as a strong inducer of ADAM17, causes not only proteolysis by ADAM17 but also a rapid increase of the mature protease at the cell surface. This is followed by internalisation and subsequent degradation of the protease. Eventually, this leads to a substantial downregulation of mature ADAM17. Our results therefore imply that physiological activation of ADAM17 does not rely on its relocalisation, but that PMA-induced PKC activity drastically dysregulates the localisation of ADAM17.