Endocytosis of the signal transducing receptor gp130 as mechanism to fine-tune IL-6 responses

Background and aims: The restriction of cytokine signalling in strength and duration is of fundamental importance to limit inflammation. We identified a cross-talk mediated endocytosis by which pro-inflammatory stimuli can influence the responsiveness of liver cells for cytokines of the IL-6 family.

Methods: HepG2 hepatoma cells were treated with the pro-inflammatory cytokine IL-1β for different periods of time. IL-1β-mediated signalling was characterized by Western blotting. Cell surface expression of gp130 was analyzed by flow cytometry and live cell imaging. To identify the signalling pathway responsible for the IL-1β-mediated endocytosis of gp130 site-directed mutagenesis and siRNA technology were used. The impact of reduced cell surface expression of gp130 on acute-phase protein expression was determined by reporter gene assay and qPCR. The in vivo relevance of this cross-talk mediated endocytosis of gp130 was addressed by the generation of a gp130 knock-in mouse line carrying a point mutation in the gp130 di-leucine internalization motif.

Results: IL-1β pretreatment of HepG2 hepatoma cells impaired IL-6-induced JAK/STAT signaling through serine phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalization and degradation. Investigation of the underlying molecular mechanism identified the activation of the p38/MK2 kinase axis as crucial step. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalization motif blocked receptor depletion and restored IL-6-dependent STAT activation as well as acute phase gene induction. The endocytosis process is mediated via clathrin-coated vesicles and therefore substantially differs from ligand-mediated endocytosis. The in vivo relevance of this novel regulatory mechanism to fine-tune IL-6 responses is currently been analyzed in newly generated endocytosis-deficient gp130 knock-in mice.

Conclusion: Our data imply differential routing of gp130 depending on the individual stimulus and cell type which might have far reaching consequences for the control of the cytokine signalling strength during inflammatory processes.

Corresponding author: Hermanns, Heike M

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