Interleukin-1 Stimulates ADAM17 through a Mechanism Independent of its Cytoplasmic Domain or Phosphorylation at Threonine 735
Figure 2
Effect of inhibitors of p38 MAPK or PKC on stimulated shedding by ADAM17T735A and ADAM17ΔC.
A) Adam17−/− mEFs were co-transfected with TGFα-AP and murine WT ADAM17, EA, T735A or ΔC mutants, and either not treated (black bars) or stimulated with IL-1β (5 ng/ml, light grey bars) or anisomycin (1 µM, dark grey bars), in the presence or absence of 10 µM of the p38 MAPK inhibitor SB203580 (SB203) as indicated, with DMSO serving as a control. Constitutive and stimulated shedding of TGFα-AP was rescued by cotransfection with WT ADAM17 as well as the T735A and ΔC mutants, but not by the inactive EA mutant. There was no significant difference between the constitutive or stimulated shedding of TGFα-AP from Adam17−/− mEFs rescued with WT compared to the T735A or ΔC mutant of ADAM17. IL-1β and anisomycin-stimulated shedding of TGFα-AP in cells rescued with WT, T735 or ΔC was blocked to a comparable extent by 10 µM of the p38 MAPK inhibitor SB203580. B) TGFα-AP shedding from Adam17−/− cells rescued with WT ADAM17, T735A or ΔC was stimulated to a comparable level by 25 ng/ml PMA (untreated, black bars, PMA-treated, grey bars), and the stimulation could be blocked by the PKC inhibitor BIM I (4 µM). C) Adam17−/− mEFs transfected with TGFα together with WT ADAM17 were stimulated with 5 ng/ml IL-1β, 1 µM anisomycin or 25 ng/ml PMA in the presence or absence of 4 µM BIM I. BIM I blocked the TGFα-AP shedding stimulated by PMA, but had no significant effect on the shedding stimulated by IL-1β or anisomycin. All results represent the average values of at least 3 experiments, and the error bars indicate +/− standard error of the mean (sem). Asterisks indicate a significant increase upon addition of a stimulus, whereas plus (+) indicates a significant decrease in stimulated shedding upon introduction of an inhibitor (students t-test, p<0.05).