Abstract
(Pro)renin receptor (PRR) contains overlapping cleavage site for site-1 protease (S1P) and furin for generation of soluble PRR (sPRR). Although S1P-mediated cleavage mediates the release of sPRR, the functional implication of furin-mediated cleavage is unclear. Here we tested whether furin-mediated cleavage was required for the activity of sPRR in activating ENaC in cultured M1 cells. M1 cells were transfected with pcDNA3.4 containing full-length PRR with (Furin Mut) or without (WT) mutagenesis of furin cleavage site; empty vector (EM) served a control. As compared with EM, overexpression of WT and Furin Mut vectors induced a more than 16-fold comparable increase in the release of sPRR. Amiloride-sensitive short circuit current as assessed by Ussing chamber technique was elevated by overexpression of WT PRR which was reduced by 37% by Furin Mut (ENaC activity: 1.00 + 0.06 μA/cm 2 in EM, 1.67 + 0.05 μA/cm 2 in WT, and 1.04 + 0.07 μA/cm 2 in Furin Mut, p < 0.05). In parallel, the expression of α-ENaC but not β or γ subunit as assessed by immunoblotting and qRT-PCR analysis was elevated by WT PRR and this increase was blunted by Furin Mut. In a separate experiment, M1 cells were transfected with pcDNA3.4 containing cDNA for sPRR with S1P cleavage (AA 1-282) (sPRR-S1P) or with furin cleavage (AA 1-279) (sPRR-furin); empty vector was used as a control. Overexpression of cDNA for the two types of sPRR induced a significant and comparable increase in the release of sPRR. By Ussing chamber technique, ENaC activity was 1.00 + 0.09 μA/cm 2 in EM, 1.03 + 0.10 μA/cm 2 in sPRR-S1P, and 1.39 + 0.14 μA/cm 2 in sPRR-furin, p < 0.05. Lastly, 293 cells were pretreated for 1 h with furin inhibitor α1-antitrypsin Portland followed by transfection with empty vector, WT PRR, or Furin Mut vectors. sPRR in the condition medium was enriched by using protein centrifugal filter devices and applied to M1 cells for 10 min followed by measurement of ENaC activity. Pretreatment with furin inhibition attenuated ENaC-acting activity induced by overexpression of WT PRR. Overall, the three independent approaches consistently demonstrated that furin-mediated modification is required for the activity of sPRR to increase ENaC-mediated Na + transport in the CD cells.
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