(Pro)renin receptor mediates both angiotensin II-dependent and -independent oxidative stress in neuronal cells

Abstract

The binding of renin or prorenin to the (pro)renin receptor (PRR) promotes angiotensin (Ang) II formation and mediates Ang II-independent signaling pathways. In the central nervous system (CNS), Ang II regulates blood pressure via inducing oxidative stress; however, the role of PRR-mediated Ang II-independent signaling pathways in oxidative stress in the CNS remains undefined. To address this question, Neuro-2A cells were infected with control virus or an adeno-associated virus encoding the human PRR. Human PRR over-expression alone increased ROS levels, NADPH oxidase activity, as well as NADPH oxidase (NOX) isoforms 2 and 4 mRNA expression levels and these effects were not blocked by losartan. Moreover, the increase in NOX 2 and NOX 4 mRNA levels, NADPH oxidase activity, and ROS levels induced by PRR over-expression was prevented by mitogen activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MAPK/ERK1/2) inhibition, and phosphoinositide 3 kinase/Akt (IP3/Akt) inhibition, indicating that PRR regulates NOX activity and ROS formation in neuro-2A cells through Ang II-independent ERK1/2 and IP3/Akt activation. Interestingly, at a concentration of 2 nM or higher, prorenin promoted Ang II formation, and thus further increased the ROS levels in cultured Neuro-2A cells via PRR. In conclusion, human PRR over-expression induced ROS production through both angiotensin II-dependent and -independent mechanisms. We showed that PRR-mediated angiotensin II-independent ROS formation is associated with activation of the MAPK/ERK1/2 and PI3/Akt signaling pathways and up-regulation of mRNA level of NOX 2 and NOX4 isoforms in neuronal cells.

Figure 1. Characterization of AAV-hPRR-eGFP in Neuro-2A cells.

Representative images (A) showing the GFP fluorescence (green), PRR immunofluorescence staining (red), nuclear staining by DAPI (blue), and the co-localization of GFP with PRR 3 d after virus infection with AAV-eGFP or AAV-hPRR-eGFP. A semi-quantitative graph of PRR immunostaining (B), a quantitative real-time PCR for mouse PRR and human PRR mRNA expression (C), and PRR and GFP protein expression by western blot (D) in Neuro-2A cells. *P<0.05 vs. AAV-eGFP.

Figure 2. PRR over-expression induces Ang II-dependent and -independent ROS production in neuronal cells.

Representative DHE staining images (A) and a semi-quantitative graph (B) for ROS levels 3 days post virus infection in Neuro-2A cells. Superoxide production (C) was measured by Electron paramagnetic resonance (EPR). * P<0.05 vs. AAV-eGFP with the same treatment, # P<0.05 vs. AAV-eGFP with vehicle. @ P<0.05vs. AAV-hPRR-eGFP with prorenin.

Figure 3. Ang II, prorenin, and renin levels in Neuro-2A cells.

Ang II levels in cell lysate (A) and cell culture medium (B) following treatment of vehicle or mouse prorenin (0.1, 1, 2, and 10 nM; 20 min), with or without captopril (10 µM; 30 min) 3 d after virus infection. Representative western blotting and semi-quantitative graph of prorenin and renin protein levels in cell lysate (C) and cell culture medium (D). For panel A and B: *P<0.05 vs. AAV-eGFP with vehicle. # P<0.05 vs. AAV-eGFP with the same treatment. & P<0.05 vs. prorenin (2 nM). @ P<0.05 vs. prorenin (10 nM). For panel C and D: * P<0.05 vs. renin.

Figure 4. PRR mediates ROS production via up-regulation of NOX2 and NOX4 levels.

NOX1, NOX2, and NOX4 mRNA levels in Neuro-2A cells (A). NOX2 (B) and NOX4 (C) mRNA levels in brain hypothalamic tissue 3 d after ICV injection of AAV-hPRR-eGFP or control virus. The knockdown efficiency of NOX2 siRNA (D) and NOX4 siRNA (E). A summary of the relative DHE fluorescence following NOX2 siRNA or NOX4 siRNA, or a combination of both (F). *P<0.05 vs. AAV-eGFP with scramble siRNA. # P<0.05 vs. scramble siRNA with the same virus.

Figure 5. PI3K, MAPK activation mediates PRR over-expression-induced NOX2 and NOX4 up-regulation and ROS production.

Figure 6. PRR over-expression activates ERK1/2 and Akt phosphorylation in Neuro-2A cells.

A representative western blot and semi- quantitative graph for ERK1/2 (A) and Akt (B) phosphorylation in the presence of wortmannin (2 µM) or U0126 (10 µM) 3 d post AAV-hPRR-eGFP or control virus infection in Neuro-2A cells. All the experiments were done in the presence of losartan (10 µM). *P<0.05 vs. AAV-eGFP in the same treatment. # P<0.05 vs. vehicle treatment.

Figure 7. The Ang II-dependent and -independent PRR signals in ROS production.

In neuronal cells, PRR over-expression activates Ang II-dependent and -independent ROS production via activation of PI3K/Akt and ERK1/2, up-regulation of NOX2 and NOX4, and subsequent increases in NADPH oxidase activity and ROS production, possibly through prorenin. Abbreviations: ROS, reactive oxygen species; PRR, (pro)renin receptor; AGT, angiotensinogen; Ang I, angiotensin I; ACE, angiotensin-converting enzyme; Ang II, angiotensin II; AT1R, angiotensin II type I receptor; NADPH oxidase, nicotinamide adenine dinucleotide phosphate-oxidase; PI3K, phosphoinositide-3-kinase; MAPK, mitogen activated protein kinase; ERK1/2, extracellular signal-regulated kinase.