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

Charlotte M Flynn,Christoph Garbers,Juliane Lokau,Christoph Becker-Pauly,Diana Dudziak,Yvonne Garbers,Christian H K Lehmann,Samadhi Aparicio-Siegmund,Bernd Schröder,Stefan Düsterhöft,Stefan Rose-John,Rielana Wichert

doi:10.1038/s41598-020-77884-4

Abstract

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.

Highlights

The cytokine interleukin-6 (IL-6) fulfills its pleiotropic functions via different modes of signaling
Initial studies described a responsible protease that was activated by the phorbol ester PMA in a protein kinase C (PKC)-dependent manner[19], which was later identified as A DAM1734,35
We determined the correct cleavage site of the IL-6 receptor (IL-6R) by ADAM17 via mass spectrometry (MS) analyses of precipitated soluble forms of the IL-6R (sIL-6R) from human serum and identified a C-terminal peptide of the sIL-6R that originated from proteolytic cleavage and ended with the residue Pro-355

Summary

Introduction

The cytokine interleukin-6 (IL-6) fulfills its pleiotropic functions via different modes of signaling. The formation of the IL-6/ IL-6R complex induces homodimerization of the common β-receptor glycoprotein (gp)[130] and subsequently the activation of several intracellular signaling cascades, e.g. the Janus kinase/Signal Transducer and Activator of Transcription (Jak/STAT), the phosphoinositide 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) pathways[6] This mode of signaling has been termed classic signaling and is thought to be responsible for the regenerative, anti-inflammatory properties of IL-63,7. A single nucleotide polymorphism (rs2228145), which results in the exchange of an asparagine into an alanine residue at position 358 of the IL-6R protein in close proximity of the ADAM17 cleavage site, results in increased sIL-6R levels[11,12] and is associated with a reduced risk of coronary heart disease[13,14] The reason for this might be an anti-inflammatory effect on an increased buffer capacity of sIL-6R in combination with soluble gp[130] (sgp130), which can bind and neutralize low levels of systemic IL-615,16. SIL-6R serum levels are unaltered in hypomorphic ADAM17ex/ex mice, suggesting that another protease besides ADAM17 is responsible for the generation of sIL-6R serum levels under physiological conditions

Methods

Results

Conclusion