Angiotensin II regulates brain (pro)renin receptor expression through activation of cAMP response element-binding protein

Abstract

We reported that brain (pro)renin receptor (PRR) expression levels are elevated in DOCA-salt-induced hypertension; however, the underlying mechanism remained unknown. To address whether ANG II type 1 receptor (AT1R) signaling is involved in this regulation, we implanted a DOCA pellet and supplied 0.9% saline as the drinking solution to C57BL/6J mice. Sham pellet-implanted mice that were provided regular drinking water served as controls. Concurrently, mice were intracerebroventricularly infused with the AT1R blocker losartan, angiotensin-converting-enzyme inhibitor captopril, or artificial cerebrospinal fluid for 3 wk. Intracerebroventricular infusion of losartan or captopril attenuated DOCA-salt-induced PRR mRNA elevation in the paraventricular nucleus of the hypothalamus, suggesting a role for ANG II/AT1R signaling in regulating PRR expression during DOCA-salt hypertension. To test which ANG II/AT1R downstream transcription factors were involved in PRR regulation, we treated Neuro-2A cells with ANG II with or without CREB (cAMP response element-binding protein) or AP-1 (activator protein-1) inhibitors, or CREB siRNA. CREB and AP-1 inhibitors, as well as CREB knockdown abolished ANG II-induced increases in PRR levels. ANG II also induced PRR upregulation in primary cultured neurons. Chromatin immunoprecipitation assays revealed that ANG II treatment increased CREB binding to the endogenous PRR promoter in both cultured neurons and hypothalamic tissues of DOCA-salt hypertensive mice. This increase in CREB activity was reversed by AT1R blockade. Collectively, these findings indicate that ANG II acts via AT1R to upregulate PRR expression both in cultured cells and in DOCA-salt hypertensive mice by increasing CREB binding to the PRR promoter.

Keywords: (pro)renin receptor; CAMP response element-binding protein; central nervous system; renin-angiotensin system; salt-sensitive hypertension.

Fig. 1.

DOCA-salt treatment increases brain PRR expression in an ANG II-dependent manner. A: PRR mRNA levels in the paraventricular nucleus (PVN) of the hypothalamus of mice in the control (SHAM) group and in mice treated with DOCA-salt for 1 day (DOCA D1), 3 days (DOCA D3), 7 days (DOCA D7), or 21 days (DOCA D21) (n = 5 or 6 PVNs/group; *P < 0.05 vs. SHAM). B: PRR mRNA levels in the PVN of mice treated with SHAM; SHAM + intracerebroventricular (ICV) infusion of losartan; or DOCA-salt with ICV infusion of artificial cerebrospinal fluid (aCSF), losartan, or captopril for 21 days (n = 5 or 6 PVNs/group; *P < 0.05 vs. SHAM, #P < 0.05 vs. DOCA + ICV aCSF D21). C: mean arterial pressure (MAP) recorded by radiotelemetry in mice treated with DOCA-salt and intracerebroventricularly infused aCSF or captopril. Arrow indicates the initiation of DOCA-salt treatment and intracebroventricular infusion (n = 5 mice/group; *P < 0.05 vs. DOCA + ICV aCSF). D: representative Western blots for PRR and β-actin, and quantification of PRR protein expression in the hypothalamus of SHAM mice and mice treated with DOCA-salt for 21 days (n = 4 hypothalami/group; *P < 0.05 vs. SHAM).

Fig. 2.

Inhibition of AT₁R, AP-1, or CREB prevents ANG II-induced increases in PRR mRNA levels in Neuro-2A cells. A: PRR mRNA levels in Neuro-2A cells incubated with or without (control) ANG II (100 nM, 1 h) in the presence or absence of losartan (10 μM) for 2 h (n = 4 in control and ANG II groups; n = 3 in control + losartan group and ANG II + losartan group). B: PRR mRNA levels in control, control + CREB inhibitor (CAS92-78-4; 10 μM), ANG II (100 nM, 1 h), and ANG II + CAS92-78-4 (n = 3) groups. C: PRR mRNA levels in control, control + AP-1 inhibitor (SR11302; 10 μM), ANG II (100 nM, 1 h), and ANG II + SR11302 (n = 3) groups. D: PRR mRNA levels in control, control + NF-κB inhibitor (parthenolid; 10 μM), ANG II (100 nM, 1 h), and ANG II + parthenolid (n = 3) groups. n values represent the number of separate experiments; within an experiment; triplicate determinations were performed for each group (*P < 0.05 vs. control. #P < 0.05 vs. ANG II).

Fig. 3.

CREB knockdown prevents ANG II-induced (pro)renin receptor (PRR) upregulation. A: representative Western blots for P-CREB, T-CREB, and β-actin, and quantification of P-CREB and T-CREB protein expression in Neuro-2A cells transfected with scrambled siRNA or CREB siRNA (n = 3; *P < 0.05 vs. scrambled siRNA). B: representative Western blots for PRR and β-actin, and quantification of PRR protein expression in Neuro-2A cells treated with ANG II (100 nM) for 2, 4, or 6 h (n = 3; *P < 0.05 vs. control). C: representative Western blots for PRR and β-actin, and quantification of PRR protein expression in Neuro-2A cells transfected with or without CREB siRNA in the presence and absence of ANG II (100 nM) stimulation for 4 h (*P < 0.05 vs. control, #P < 0.05 vs. ANG II; n = 3). D: PRR mRNA levels in control, control + CREB siRNA, ANG II (100 nM for 1 h), and ANG II + CREB siRNA (n = 4) groups (*P < 0.05 vs. control, #P < 0.05 vs. ANG II). n values represent the number of separate experiments; within an experiment, duplicate (for protein) or triplicate (for mRNA) determinations were performed for each group.

Fig. 4.

ANG II increases PRR expression in primary cultured neurons. A: DIC (gray) imaging and DAPI (cyan) counterstaining of primary neurons (A1), immunostaining with the neuronal marker MAP2 (red) (A2), and merged image of MAP2, DAPI, and DIC (×64 magnification) (A3). B: PRR mRNA levels in controls and animals treated with ANG II (100 nM) for 1, 2, or 4 h (n = 3; *P < 0.05 vs. control). C: representative Western blots for PRR and β-actin and quantification of PRR protein expression in primary cultured neurons treated with ANG II (100 nM) for 2, 4, or 6 h (n = 3; *P < 0.05 vs. control). n values represent the number of separate experiments; within an experiment, duplicate (for protein) or triplicate (for mRNA) determinations were performed for each group.

Fig. 5.

ANG II increases CREB and c-Jun phosphorylation in Neuro-2A cells. A: representative Western blots for P-CREB, T-CREB, and β-actin in Neuro-2A cells at different time points after treatment with ANG II (100 nM) or ANG II + losartan for 60 min. B: quantification of P-CREB following ANG II treatment. C: quantification of T-CREB following ANG II treatment. D: representative Western blots for P-c-Jun, T-c-Jun, and β-actin at different time points after treatment with ANG II or ANG II + losartan for 60 min. E: quantification of P-c-Jun following ANG II treatment. F: quantification of T-c-Jun following ANG II treatment (n = 3; *P < 0.05 vs. control, #P < 0.05 vs. ANG II treatment). n values represent the number of separate experiments; within an experiment, duplicate determinations were performed for each group.

Fig. 6.

ChIP assays showing ANG II-induced increases in CREB binding to the endogenous PRR promoter in Neuro-2A cells. A: design of primers targeting the PRR promoter, covering a 1,042-bp region upstream of the starting codon. B: ChIP assay results showing relative enrichment for P-CREB binding to the PRR promoter in control and ANG II-treated cells. C: relative enrichment for P-c-Jun binding to the PRR promoter in control and ANG II-treated cells (n = 3; *P < 0.05 vs. control). n values represent the number of separate experiments; within an experiment, triplicate determinations were performed for each group.

Fig. 7.

CREB phosphorylation is increased in the hypothalamus of DOCA-salt hypertensive mice. A: representative Western blots for P-CREB, T-CREB, and β-actin in the hypothalamus of mice in SHAM, SHAM + ICV losartan, DOCA-salt, and DOCA-salt + ICV-infused losartan (3 mg·kg⁻¹·day⁻¹) groups after 21 days of treatment. B: quantification of P-CREB following treatment with DOCA-salt, with or without intracerebroventricular infusion of losartan. C: quantification of T-CREB following treatment with DOCA-salt, with or without intracerebroventriclar infusion of losartan (n = 4 or 5 hypothalami/group; *P < 0.05 vs. SHAM, #P < 0.05 vs. DOCA + ICV aCSF).

Fig. 8.

An ANG II/AT₁R-dependent increase in CREB binding to the endogenous PRR promoter is responsible for PRR upregulation during DOCA-salt hypertension. A: ChIP assay showing relative enrichment for P-CREB binding to the PRR promoter in the hypothalamus of mice in SHAM, SHAM + ICV losartan, DOCA-salt, and DOCA-salt + ICV-infused losartan (3 mg·kg⁻¹·day⁻¹) groups after 21 days of treatment (n = 5 hypothalami/group; *P < 0.05 vs. control, #P < 0.05 vs. DOCA + ICV aCSF). B: PRR mRNA levels in the PVN of mice in SHAM, SHAM + ICV infusion of losartan, and DOCA-salt with ICV infusion of aCSF or losartan groups after 21 days of treatment (n = 4 or 5 PVNs/group; *P < 0.05 vs. SHAM, #P < 0.05 vs. DOCA + ICV aCSF).