This study is about (pro)renin receptor (PRR) is a multifunctional integral membrane protein that serves as a component of the vacuolar H+-ATPase (V-ATPase) and also activates (pro)renin. We recently showed that full-length PRR, found as part of a V-ATPase sub-complex, is abundant in extracellular vesicles shed by osteoclasts. Here, we tested whether these extracellular vesicles stimulate (pro)renin. Extracellular vesicles isolated from the conditioned media of RAW 264.7 osteoclast-like cells or primary osteoclasts were characterized and counted by nanoparticle tracking. Immunoblotting confirmed that full-length PRR was present. Extracellular vesicles from osteoclasts dose-dependently stimulated (pro)renin activity, while extracellular vesicles from 4T1 cancer cells, in which we did not detect PRR, did not activate (pro)renin.
FIG: Characterization of EVs from RAW 264.7 osteoclast-like cells and primary mouse osteoclasts. RAW 264.7 cells or primary hematopoietic cells were stimulated to differentiate into osteoclasts. (A) RAW 264.7 osteoclast-like cells stained for TRAP activity (Pink is positive). Scale bar = 50 microns. (B) Primary osteoclasts stained for TRAP activity. Scale bar 50 microns. (C) Nanoparticle tracking of EVs collected from the conditioned media of RAW 264.7 osteoclast-like cells. (D) Nanoparticle tracking data of EVs collected from conditioned media of primary osteoclasts. The black track in the plots is the mean number for each size from 5 different 60 s trials, the red indicates the standard error boundaries at each size. The concentration of particles reflects the diluted concentration of the isolated EVs. Mean, and mode reflect the data from the particle size of the 5 trials. Standard Deviation reflects the spread of the data. D10, D50, and D90 indicates the size where 10, 50 or 90 percent of the detected particles are smaller, and reflects another measure of the spread of the data.
To confirm that the ability of extracellular vesicles from osteoclasts to stimulate (pro)renin activity was due to the PRR, the “handle region peptide” from the PRR, a competitive inhibitor of PRR activity, was tested. It dose-dependently blocked the ability of extracellular vesicles to stimulate the enzymatic activity of (pro)renin. In summary, the PRR, an abundant component of extracellular vesicles shed by osteoclasts, stimulates (pro)renin activity. This represents a novel mechanism by which extracellular vesicles can function in intercellular regulation, with direct implications for bone biology.
Murray, J.B., Mikhael, C., Han, G. et al. Activation of (pro)renin by (pro)renin receptor in extracellular vesicles from osteoclasts. Sci Rep11, 9214 (2021). https://doi.org/10.1038/s41598-021-88665-y