Table 1.
Primer and TaqMan probe sequences for real time PCR.
Fig 1.
Effects of RXR pan-agonist PA024 on cell proliferation/apoptosis, CYP11B2 mRNA expression/promoter activity, and aldosterone secretion in H295R cells.
Effects of PA024 on proliferation and apoptosis are shown in (A) and (B), respectively. H295R cells were incubated for 48 h either in the presence (100 nmol/L, 1 μmol/L, 5 μmol/L, 10 μmol/L) or absence (control) of PA024, before each assay. Results are expressed as percentages of each control. Data represent mean ± S.E.M. (n = 6). n.s.: not significant versus control. (C), total RNAs extracted from the cells treated with PA024 (the indicated concentration, 24 h) and Ang II (100 nmol/L, 6 h) were examined for CYP11B2 mRNA expression by quantitative real-time PCR. (D), H295R cells transiently transfected for 24 h with CYP11B2-luc (-1521 to +2-luc) and pCMV-β-gal were treated with PA024 (indicated concentrations, 24 h) and Ang II (100 nmol/L, 6 h), before the luciferase assay. Data represent mean ± S.E.M. (n = 4), percent of control. (E) and (F), supernatants obtained from the cells treated with PA024 (10 μmol/L, 48 h) and Ang II (100 nmol/L, 24 h) were examined for aldosterone and cortisol secretion by EIA, respectively. (E), the experiments were performed twice, and each data set (n = 4) was pooled and shown as one data set (n = 8). Data represent mean ± S.E.M. (n = 8), percent of control. (F), data represent mean ± S.E.M. (n = 4), percent of control. ***P<0.001, **P<0.01 vs. control, †P<0.05, n.s.: not significant vs. Ang II.
Fig 2.
Effects of PA024 on the CYP11B2 promoter deletion mutants and point mutants.
(A), effect of PA024 on the CYP11B2 promoter deletion mutants. Either -1521/+2-luc, -747/+2-luc, -135/+2-luc, -106/+2-luc, -65/+2-luc, or pGL3-Basic (control plasmid) was transiently transfected for 48 h with pCMV-β-gal into H295R cells, and the cells were thereafter treated with PA024 (10 μmol/L, 24 h) and Ang II (100 nmol/L, 6 h). Data represent mean ± S.E.M. (n = 4), percent of -1521/+2-luc control. (B), effect of PA024 on the CYP11B2 promoter point mutants. Either -1521/+2-luc, -1521/+2-luc-NBRE-1-mut, -1521/+2-luc-Ad5-mut, or -1521/+2-luc-Ad1/CRE-mut was transiently transfected for 48 h with pCMV-β-gal into H295R cells, and the cells were thereafter treated with PA024 (10 μmol/L, 24 h) and Ang II (100 nmol/L, 6 h). Data represent mean ± S.E.M. (n = 4), percent of -1521/+2-luc control. ***P<0.001, **P<0.01 vs. Ang II.
Fig 3.
Possible involvement of NURR1 in the PA024-mediated suppression of CYP11B2 promoter activity via binding to the Ad5 element.
(A) and (B), dose-response analyses of the NURR1 and NGFIB mRNA expression. H295R cells were treated with PA024 (indicated concentrations, 24h) and Ang II (100 nmol/L, 6 h). Data represent mean ± S.E.M. (n = 4), percent of control. ***P<0.001 vs. control, †P<0.05 vs. Ang II. (C) and (D), effect of NURR1 or NGFIB overexpression on the CYP11B2 promoter activity. H295R cells transiently transfected with NURR1-pcDNA3, NGFIB-pcDNA3 or pcDNA3 (Mock) for 48 h were treated with PA024 (10 μmol/L, 24 h) and Ang II (100 nmol/L, 6 h), respectively. Data represent mean ± S.E.M. (n = 4), percent of pcDNA control. ***P<0.001, **P<0.01 vs. Ang II. (E), effect of PA024 on NURR1 protein expression. H295R cells were treated with PA024 (10 μmol/L, 24 h) and Ang II (100 nmol/L, 6 h). Upper panel, NURR1 protein expression indicated by arrow. Lower panel, actin protein expression indicated by arrow.
Fig 4.
Effects of PA024 on mRNA expression of other enzymes involved in steroid synthesis.
(A)-(G), total RNAs extracted from the cells treated with PA024 (10 μmol/L, 24 h) and Ang II (100 nmol/L, 6 h) were examined for mRNA expression of StAR, CYP11A1, HSD3β1, HSD3β2, CYP21A2, CYP17A1 and CYP11B1 by quantitative real-time PCR. Data represent mean ± S.E.M. (n = 4), percent of control, normalized by β-actin mRNA levels. ***P<0.001 vs. control, †P<0.05 vs. Ang II.
Fig 5.
Involvement of RXRα in the suppressive effect of PA024 on CYP11B2 mRNA expression and promoter activity.
(A) and (B), effect of RXRα knockdown by its siRNA on the mRNA expression of CYP11B2 and NURR1. H295R cells transiently transfected with siRNA (negative control or RXRα) for 48 h were incubated either in the presence (10 μmol/L) or absence (control) of PA024 for 24 h and co-treated with 100 nmol/L Ang II for the last 6 h. Results are expressed as percentages of each Ang II. Data represent mean ± S.E.M. (n = 4). **P<0.01, *P<0.05 vs. negative control siRNA at 10 μmol/L PA024. (C), effect of RXRα overexpression on the CYP11B2 mRNA expression. H295R cell transiently transfected with RXRα-pcDNA1/Amp (mRXRα) or pcDNA3 (Mock) for 48 h were incubated either in the presence (10 μmol/L) or absence (control) of PA024 for 24 h and co-treated with 100 nmol/L Ang II for the last 6 h. Results are expressed as percentages of each Ang II. Data represent mean ± S.E.M. (n = 4). *P<0.05 vs. pcDNA3 at 10 μmol/L PA024. (D), effect of RXRα overexpression on the CYP11B2 promoter activity. H295R cell transiently transfected with -1521/+2-luc, pCMV-β-gal, and RXRα-pcDNA1/Amp (mRXRα) or pcDNA3 (Mock) for 48 h were incubated either in the presence (10 μmol/L) or absence (control) of PA024 for 24 h and co-treated with 100 nmol/L Ang II for the last 6 h. Results are expressed as percentages of each Ang II. Data represent mean ± S.E.M. (n = 4). ***P<0.001 vs. pcDNA3 at 10 μmol/L PA024.
Fig 6.
Effects of the combination of pioglitazone and PA024 on CYP11B2 mRNA expression in H295R cells.
Total RNAs extracted from the cells treated with combinations of pioglitazone (10 μmol/L, 24 h), PA024 (10 μmol/L, 24 h), and Ang II (100 nmol/L, 6 h) were examined for CYP11B2 mRNA expression by quantitative real-time PCR. Data represent mean ± S.E.M. (n = 4), percent of control, normalized by β-actin mRNA levels. ***P<0.001 vs. Ang II, †††P<0.001, ††P<0.01 vs. 10 μmol/L pioglitazone + 10 μmol/L PA024
Fig 7.
Effects of PA024 on body weight, heart rate, and blood pressure in vivo.
Nine-week-old Tsukuba hypertensive mice (THM; hRN8-12 x hAG2-5), which present excessive Ang II production and chronic hypertension, were randomized for injection with either vehicle (corn oil) or PA024 (10 mg/kg/day) intraperitoneally 3 times a week for 7 weeks. Body weight (A), heart rate (HR) (B), systolic blood pressure (SBP) (C) and diastolic blood pressure (DBP) (D) were monitored once a week throughout the treatment. Data represent mean ± S.E.M. (n = 5). **P<0.01, *P<0.05, n.s: not significant vs. control mice at each week-old.
Fig 8.
Effects of PA024 on intracellular calcium ion concentration in H295R cells determined by Fluo4-AM.
H295R cells were treated with or without PA024 (10 μmol/L, 24 h). After loading with Fluo4-AM, cells were treated with 100 nmol/L Ang II, and the fluorescence change was monitored. Data represent mean (the left panel, n = 6) or mean ± S.E.M. (the right panel, n = 6), fluorescence change from time 0, arbitrary units. *P<0.05 versus Ang II.