MDM2: a novel mineralocorticoid-responsive gene involved in aldosterone-induced human vascular structural remodeling

Abstract

Aldosterone has been demonstrated to play an important role in the pathogenesis of various cardiovascular diseases. Vascular structural remodeling, including vascular smooth muscle cell (VSMC) proliferation, has been also reported in small resistance arteries of patients with primary aldosteronism. Therefore, in this study, we examined whether genes involved in the regulation of the cell cycle were induced by aldosterone alone in cultured human VSMCs and in human small resistance arteries. Results of these studies eventually demonstrated that MDM2, one of the genes involved in anti-apoptosis and cell growth, was markedly increased in mineralocorticoid receptor (MR)-positive VSMCs by aldosterone in all microarray, reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence analyses. In addition, an analysis using small interfering RNA demonstrated that this gene product was involved in cell proliferation of VSMCs induced by aldosterone. Eplerenone, a specific MR antagonist, inhibited this gene induction by aldosterone in VSMCs. MDM2 protein was also more abundant in VSMCs of small resistance arteries in patients with primary aldosteronism compared with a control population. MDM2 is therefore considered one of the mineralocorticoid-responsive genes that regulates cell proliferation of VSMCs induced by MR-mediated aldosterone stimulation, possibly playing an important role in aldosterone-induced vascular structural remodeling.

Figure 1-6919

A: Representative RT-PCR analysis of total RNA from cultured human VSMCs (HASMC). RNA was amplified in the presence of oligonucleotide primers specific for MR (top panel) and 11β-HSD type 2 (bottom panel). Extracts from human kidney were used as a positive control (P). B: Representative immunoblotting studies demonstrating MR and 11β-HSD type 2 proteins in HASMC. Extracts from human kidney were used as a positive control (P). C and D: Immunocytochemical analysis demonstrated that immunopositive cells for MR appear brown as a result of diaminobenzidine colorimetric reaction in HASMC without ligand stimulation (C) or treated with 10 nmol/L aldosterone (D) (Original magnification, ×400).

Figure 2-6919

Results of RT/real-time PCR analysis for MDM2 in HASMC among cells treated with vehicle (0.1% ethanol) (control), aldosterone alone (100 pmol/L, 10 nmol/L), aldosterone (10 nmol/L) with eplerenone (100 nmol/L), aldosterone (10 nmol/L) with CHX, and aldosterone (10 nmol/L) with ACD (100 nmol/L), respectively, after 8 hours. Data are shown as mean ± SD (*P < 0.05).

Figure 3-6919

A: Representative immunoblotting studies demonstrating MDM2 proteins in HASMC among cells treated with vehicle (0.1% ethanol) (control), aldosterone alone (10 nmol/L), and aldosterone (10 nmol/L) with eplerenone (100 nmol/L), respectively, after 48 hours. B: Relative levels of MDM2 protein expression in HASMC among cells treated with vehicle (0.1% ethanol) (control), aldosterone alone (10 nmol/L), and aldosterone (10 nmol/L) with eplerenone (100 nmol/L), respectively, after 48 hours. Data are shown as mean ± SD (*P < 0.05).

Figure 4-6919

Representative immunofluorescence studies demonstrating MDM2 proteins in HASMC among cells treated with vehicle (0.1% ethanol) (control) (A), aldosterone alone (10 nmol/L) (B), and aldosterone (10 nmol/L) with eplerenone (100 nmol/L) (C), respectively, after 48 hours. MDM2 protein expression (green) was remarkably abundant in the nucleus of cells treated by aldosterone using fluorescein isothiocyanate (FITC)-conjugated secondary antibody (B), and its expression was blocked by eplerenone (C). 4,6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining (blue).

Figure 5-6919

A: Expression of MDM2, GAPDH, and RPL13A mRNAs at 24 hours after the transfection of siRNA (0, 10, or 100 nmol/L) against MDM2 and/or GAPDH in HASMC cells detected by real-time PCR, respectively. RPL13A expression was monitored as the control. The ratio of MDM2 to RPL13A and/or GAPDH to RPL13A was calculated, and values were normalized as the ratio at no treatment (0 nmol/L), respectively. B: The levels of cell numbers in HASMC at 48 hours after treatment with vehicle (0.1% ethanol) (control) or aldosterone (10 nmol/L) after transfection of MDM2 siRNA (0, 10, or 100 nmol/L) or under the presence of eplerenone (100 nmol/L). Data are presented as mean ± SD (*P < 0.05). C: The levels of cell numbers in HASMC at 72 hours after treatment with vehicle (0.1% ethanol) (control) or aldosterone (10 nmol/L) after transfection of MDM2 siRNA (0, 10, or 100 nmol/L) or under the presence of eplerenone (100 nmol/L). Data are represented as mean ± SD (*P < 0.05). N.S., not significant.

Figure 6-6919

A–C: Immunohistochemistry for MDM2 in VSMCs of small resistance arteries adjacent to adrenal tumors or normal adrenal glands. Immunoreactivity for MDM2 in VSMCs of small resistance arteries adjacent to adrenal tumor of primary aldosteronism (A), nonfunctioning adrenal tumor (B), and non-neoplastic adrenal gland (C). Immunoreactivity appears brown as a result of diaminobenzidine colorimetric reaction. Original magnification, ×400. D: The relative immunoreactivity was evaluated by the LI in each group (0 to 100). Data are represented as mean ± SD (*P < 0.05). PA, aldosterone-producing adrenocortical adenoma; NF, nonfunctioning tumor; normal, non-neoplastic adrenal gland.