Production of soluble Neprilysin by endothelial cells.
A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35±0.70 and 6.54±0.41 μmols of substrate cleaved over 3h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC50 values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37±1.43 and 38.30±4.70, respectively as a % of control; P<0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.
Department of Biochemistry & Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800, Australia. Electronic address: Sanjaya.Kuruppu@monash.edu.
Sanjaya
S
Kuruppu
Department of Physiology, Building 13F, Monash University, Wellington Rd, Clayton, Vic 3800, Australia.
Niwanthi W
NW
Rajapakse
Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, FL 34987, USA.
Dmitriy
D
Minond
Department of Biochemistry & Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800, Australia.
A Ian
AI
Smith
D051722:ADAM Proteins / Q000378:metabolism*; D000072198:ADAM17 Protein; D002384:Catalysis; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism*; D055354:Exosomes / Q000378:metabolism*; D006801:Humans; D015260:Neprilysin / Q000096:biosynthesis* / Q000737:chemistry; D015398:Signal Transduction / Q000502:physiology; D012995:Solubility
D051722:ADAM Proteins; D015260:Neprilysin; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human
ADAM; Endothelial cell; Enzymes; Exosomes; Shedding