Open AccessFunctional and molecular evidence for expression of the renin angiotensin system and ADAM17-mediated ACE2 shedding in COS7 cells
Author(s) -
Nadja Grobe,
Mauricio Di Fulvio,
Nada Kashkari,
Harshita Chodavarapu,
Hari K. Somineni,
Richa Singh,
Khalid M. Elased
Publication year - 2015
Publication title -
american journal of physiology. cell physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.432
H-Index - 181
eISSN - 1522-1563
pISSN - 0363-6143
DOI - 10.1152/ajpcell.00247.2014
Subject(s) - ectodomain , renin–angiotensin system , endogeny , cell culture , microbiology and biotechnology , receptor , angiotensin ii , biology , angiotensin converting enzyme 2 , cell , western blot , small hairpin rna , metalloproteinase , chemistry , enzyme , endocrinology , medicine , biochemistry , gene , gene knockdown , genetics , disease , covid-19 , blood pressure , infectious disease (medical specialty)
The renin angiotensin system (RAS) plays a vital role in the regulation of the cardiovascular and renal functions. COS7 is a robust and easily transfectable cell line derived from the kidney of the African green monkey, Cercopithecus aethiops. The aims of this study were to 1) demonstrate the presence of an endogenous and functional RAS in COS7, and 2) investigate the role of a disintegrin and metalloproteinase-17 (ADAM17) in the ectodomain shedding of angiotensin converting enzyme-2 (ACE2). Reverse transcription coupled to gene-specific polymerase chain reaction demonstrated expression of ACE, ACE2, angiotensin II type 1 receptor (AT1R), and renin at the transcript levels in total RNA cell extracts. Western blot and immunohistochemistry identified ACE (60 kDa), ACE2 (75 kDa), AT1R (43 kDa), renin (41 kDa), and ADAM17 (130 kDa) in COS7. At the functional level, a sensitive and selective mass spectrometric approach detected endogenous renin, ACE, and ACE2 activities. ANG-(1-7) formation (m/z 899) from the natural substrate ANG II (m/z 1,046) was detected in lysates and media. COS7 cells stably expressing shRNA constructs directed against endogenous ADAM17 showed reduced ACE2 shedding into the media. This is the first study demonstrating endogenous expression of the RAS and ADAM17 in the widely used COS7 cell line and its utility to study ectodomain shedding of ACE2 mediated by ADAM17 in vitro. The transfectable nature of this cell line makes it an attractive cell model for studying the molecular, functional, and pharmacological properties of the renal RAS.