FIG. 6.

ROS increase the levels of expression and activity of ADAM9 in mEFs. (A) Wild-type mEFs (50 to 60% culture density) were treated with 50 to 200 μM of H₂O₂ for 6 h, and ADAM9 was detected by Western blotting (bottom). Treatment with H₂O₂ increased the level of expression of ADAM9 in wild-type mEFs. (A and B) Wild-type (A) and Adam9^−/− (B) mEFs were transfected with EphB4 as a readout for the catalytic activity of endogenous ADAM9 and treated with increasing concentrations of H₂O₂. Increased levels of shedding of EphB4 were seen in an H₂O₂ concentration-dependent manner in wild-type cells (A, top) but not in Adam9^−/− cells (B), providing the first evidence for processing of a substrate by endogenously upregulated ADAM9 in wild-type cells. (C) Wild-type (WT) mEFs transfected with EphB4 were either not treated or treated with 200 μM H₂O₂, with 1 μg/ml actinomycin D (Act-D), or with both 200 μM H₂O₂ and 1 μg/ml actinomycin D. Actinomycin D prevented the increase in levels of EphB4 shedding from cells treated with H₂O₂ (top) and the increase in ADAM9 protein levels (bottom), suggesting that H₂O₂ enhances the transcription of ADAM9. (D) Levels of shedding of EphB4 from Adam9^−/− cells can be increased by cotransfection with wild-type ADAM9, but there is no further enhancement by treatment with H₂O₂ (50 to 200 μM). This suggests that ADAM9 is not directly activated by ROS, since the ADAM9 expression plasmid lacks putative ROS transcriptional regulatory elements. (E) Shedding of TNF-α was used to test whether its sheddase, ADAM17, responds to H₂O₂ treatment. Phorbol myristate acetate stimulated the shedding of TNF-α in wild-type mEFs, and this is known to depend on ADAM17 (28), whereas increasing concentrations of ROS did not stimulate the shedding of TNF-α in these cells. These results suggest that ADAM9 is the main sheddase responding to ROS treatment for 6 h in mEF cells.