Figure 3.

Overexpressed ADAM17 affects ACE2 levels. A, ACE2 and ADAM17 mRNA concentrations were determined from four experiments in which 832/13 cells were transfected with 0.5 μg/well mACE2/pcDNA3.1 and cotransfected with 2 μg/well of pAd17, pAd17E406A (an expression plasmid for inactive ADAM17), or pcDNA3.1(−). B–F, In six experiments, 832/13 cells were transfected with 4, 20, 100, or 500 ng mACE2/pcDNA3.1 per well and cotransfected with 2 μg/well of pAd17, pAd17E406A, or pcDNA3.1(−). We measured the activity (SensoLyte activity) with a SensoLyte ADAM17 activity kit (B), the activity by a more specific ADAM17 assay (C), the shed ACE2 activity (D), and the cellular ACE2 activity (E) compared with the average ACE2 activity level from five wild-type mice. We calculated the ratio of total shed ACE2 activity to total cellular ACE2 (F). G, The total cellular ACE2 and ADAM17 activities were determined at the beginning and end of a 24-hour incubation period for 832/13 cells that were cotransfected four times with 25 ng mACE2/pcDNA3.1 and 2 μg pAd17 per well. H, 832/13 cells were individually transfected with 25 ng/well mACE2/pcDNA3.1, 2 μg/well pAd17, and 2 μg/well pAd17E406A. The next day, cells were trypsinized and mixed for coculture of ACE2-expressing cells and cells overexpressing either active or inactive ADAM17. We measured ADAM17 activity, cellular ACE2 activity, and shed ACE2 activity. I, The control strengths of the amount of the ACE2 expression plasmid mACE2/pcDNA3.1 on cellular and shed ACE2 are determined as the slopes of the indicated regression lines for the natural logarithms of ACE2 activities plotted against the natural logarithms of the amount of mACE2/pcDNA3.1. Data were analyzed by an ANOVA (A–F) and t tests (H). Due to the large range in SDs for ACE2 activity measurements occurring with the different levels of the ACE2 expression plasmids, separate ANOVAs for the ACE2 parameters were conducted for each level of mACE2/pcDNA3.1(−) (D–F). *, **, ***, P < .05, P < .01, P < .001 vs. pAd17E406A.