Constitutive CX3CL1 cleavage is enhanced in COS-7 cells overexpressing ADAM10. (A) MP inhibitors differentially restore constitutive and PMA-inducible CX3CL1 shedding in COS-7 cells: COS-7 cells transiently transfected with CX3CL1 were treated with various concentrations of the mixed ADAM10/TACE inhibitor GW280264X or the selective ADAM10 inhibitor GI254023X and subsequently stimulated with 200 ng/mL PMA or left unstimulated. After 1 hour of incubation conditioned media were harvested and the concentration of soluble CX3CL1 was determined by ELISA (mean and SD, n = 3). (B) Overexpression of ADAM10 protein in COS-7 cells. Cells were transiently transfected with bovine HA-tagged ADAM10 or empty vector (vehicle). Subsequently, cells were lysed in SDS sample buffer and analyzed by Western blotting using a specific antiserum against the HA-tag. (C) CX3CL1 cleavage in COS-7 cells overexpressing wild-type (WT) ADAM10 or dominant-negative (DN) ADAM10 (Glu384Ala): COS-7 cells were cotransfected with CX3CL1 and either HA-tagged WT ADAM10, DN ADAM10, or empty vector. After 1 hour of incubation in the presence or absence of 200 ng/mL PMA, the concentration of released CX3CL1 in the conditioned media was determined by ELISA. Results are presented as mean and SD of triplicates performed in one experiment. Compared with the vehicle control, transfection of WT ADAM10 significantly increased CX3CL1 shedding, whereas transfection of DN ADAM10 significantly reduced the cleavage of the chemokine (P < .05 for both effects, indicated by the asterisks). The increase in shedding due to PMA stimulation (determined as difference in shedding between unstimulated and PMA-stimulated cells) was not significantly altered by transfection with either construct (P < .05, not indicated). The results shown in panels A-C are representative for 3 independent experiments.