The mechanism of cleavage of EGFR ligands induced by inflammatory cytokines in gastric cancer cells

Abstract Background & Aims: The epidermal growth factor (EGF) receptor (EGFR) can be transactivated by many factors including G-protein-coupled receptor agonists and cytokines. Although this EGFR transactivation reportedly requires a disintegrin and metalloproteinase (ADAM) that sheds the ectodomain of EGFR ligands, the detailed mechanisms are still unknown. This study evaluated the mechanism of interleukin (IL)-8- and IL-1β-dependent shedding of the EGFR ligand in KATO III cells. Methods: We established transfectants stably expressing alkaline phosphatase-tagged heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor α, or amphiregulin precursors, and depleted ADAM proteins, using short interfering RNA against ADAM10, 12, or 17. We assessed shedding of EGFR ligands by measuring AP activities in the conditioned media after IL-1β or IL-8 stimulation. EGFR activation was examined by immunoprecipitation and Western blotting using antiphosphotyrosine antibody. KB-R7785 and anti-IL-8 neutralizing antibody were used to inhibit activities of ADAMs and IL-8 action, respectively. Results: IL-8 dose dependently released the EGFR ligands and transiently phosphorylated EGFR, with a peak at 15 minutes. KB-R7785 completely blocked IL-8-induced shedding and EGFR transactivation. Depletion of ADAM10 also dramatically reduced IL-8-induced shedding and EGFR transactivation, but depletion of ADAM12 and 17 did not. IL-1β dose dependently enhanced shedding of HB-EGF, which was not blocked by KB-R7785 in the early phase. In the late phase, however, the EGFR transactivation was blocked by KB-R7785 and abrogated by anti-IL-8 neutralizing antibody. Conclusions: IL-8 induces shedding of EGFR ligands because of an ADAM10-dependent pathway in gastric cancer cells, whereas IL-1β acts principally by an ADAM-independent pathway. IL-1β-dependent prolonged EGFR transactivation involves multiple pathways, including an IL-8-dependent pathway.