Abstract 314: Evidence for the Expression of Renin Angiotensin System (RAS) and a Disintegrin and Metalloproteinase (ADAM) 17-mediated shedding of ACE2 in COS-7 cells
The renin angiotensin system (RAS) plays a vital role in the regulation of the cardiovascular and renal system. COS-7 is a robust and easily transfectable cell line derived from the kidney of the African green monkey, Cercopithecus aethiops. The aim of this study was to 1) demonstrate the presence of an endogenous and functional RAS in COS7 cells and 2) to investigate the role of ADAM17 in the ectodomain shedding of ACE2. COS-7 cells were grown to 90% confluency followed by incubation in serum-free media for 24 hours. Western blot, immunohistochemistry and mass spectrometry (MS)-based enzyme assays were used to study RAS protein expression and activity in COS7 cells. Western blot and immunostaining confirmed endogenous expression of ACE (195 kDa), ACE2 (75-70 kDa), AT1R (43 kDa), renin (41 kDa) and ADAM17 (130 kDa) in COS7 cells. A sensitive and selective MS approach was used to determine endogenous RAS enzymatic activity in COS-7 lysate and media using the natural RAS substrate Ang II (m/z 1046). MS analysis detected Ang-(1-7) formation (m/z 899) in lysate and media of COS7 cells. While Ang-(1-7) formation in media was completely blocked by the ACE2-specific inhibitor MLN-4760, Ang-(1-7) formation in the lysate was significantly inhibited by the prolyl carboxypeptidase-specific inhibitor Z-pro-prolinal. Using short-hairpin (sh) RNA-mediated technology, ADAM17 protein expression and activity was significantly reduced in silenced cells compared to normal cells. Western blot analysis showed a significant increase of ACE2 protein expression in the lysate of silenced cells compared to normal cell and ACE2 shedding into the media was significantly reduced in silenced cells. This is the first study to demonstrate endogenous expression of the RAS and ADAM17 in COS-7 cells. Use of COS-7 could enhance our understanding of the cross talk between different arms of the RAS and dissect the direct effects of ACE inhibitors and Ang II receptor blockers. The transfectable nature of this cell line makes it an attractive in vitro cell model for studying the molecular, functional and pharmacological properties of the renal RAS. The data support the utility of COS-7 for the study of ectodomain shedding of ACE2 in vitro.
Author Disclosures: N. Kashkari: None. N. Grobe: None. H. Chodavarapu: None. H.K. Somineni: None. R. Singh: None. M. Di Fulvio: None. K.M. Elased: None.
This research has received full or partial funding support from the American Heart Association, National Center.
- © 2014 by American Heart Association, Inc.