Regulation of endopeptidases EC3.4.24.15 and EC3.4.24.16 in vascular endothelial cells by cyclic strain: role of Gi protein signaling.
OBJECTIVE
Endopeptidase EC3.4.24.15 (EP24.15)- and EC3.4.24.16 (EP24.16)-specific peptide hydrolysis plays an important role in endothelium-mediated vasoregulation. Given the significant influence of hemodynamic forces on vascular homeostasis and pathology, we postulated that these related peptidases may be mechanosensitive. The objective of this study, therefore, was to investigate the putative role of cyclic strain in regulating the expression and enzymatic activity of EP24.15 and EP24.16 in bovine aortic endothelial cells (BAECs).


RESULTS
BAECs were cultured under conditions of defined cyclic strain (0% to 10% stretch, 60 cycles/min, 0 to 24 hours). Strain significantly increased EP24.15 and EP24.16 soluble activity in a force- and time-dependent manner, with elevations of 2.3+/-0.4- and 1.9+/-0.3-fold for EP24.15 and EP24.16, respectively, after 24 hours at 10% strain. Pharmacological agents and dominant-negative G protein mutants used to selectively disrupt Gi(alpha)- and Gbetagamma-mediated signaling pathways attenuated strain-dependent (24 hours, 5%) increases for both enzymes. Differences in the inhibitory profile for both enzymes were also noted, with EP24.15 displaying greater sensitivity to Gi(alpha2/3) inhibition and EP24.16 exhibiting greater sensitivity to Gi(alpha1/2) and Gbetagamma inhibition. Cyclic strain also increased levels of secreted EP24.15 and EP24.16 activity by 2.6+/-0.02- and 3.6+/-0.2-fold, respectively, in addition to mRNA levels for both enzymes (EP24.15 +42%, EP24.16 +56%).


CONCLUSIONS
Our findings suggest that cyclic strain putatively regulates both the mRNA expression and enzymatic function of EP24.15 and EP24.16 in BAECs via alternate Gi protein signaling pathways.
Cotter
Eoin J
EJ
Vascular Health Research Centre, Faculty of Science and Health, Dublin City University, Glasnevin, Dublin, Ireland.
von Offenberg Sweeney
Nicholas
N
Coen
Paul M
PM
Birney
Yvonne A
YA
Glucksman
Marc J
MJ
Cahill
Paul A
PA
Cummins
Philip M
PM
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured / Q000201:enzymology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics; D042783:Endothelial Cells / Q000201:enzymology* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D004790:Enzyme Induction; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D044387:GTP-Binding Protein beta Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D044388:GTP-Binding Protein gamma Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics; D052250:Nitric Oxide Synthase Type III; D010507:Periodicity; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction; D013314:Stress, Mechanical; D051552:beta-Adrenergic Receptor Kinases
C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D012333:RNA, Messenger; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; C062316:neurolysin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go