Differential effects of high and low steroidogenic factor-1 expression on CYP11B2 expression and aldosterone production in adrenocortical cells

Abstract

Steroidogenic factor-1 (SF-1/Ad4BP/NR5A1) plays a major role in regulating steroidogenic enzymes. We have previously shown that SF-1 inhibits aldosterone synthase (CYP11B2) reporter gene activity. Herein, we used the H295R/TR/SF-1 adrenal cells that increase SF-1 in a doxycycline-dependent fashion. Cells were incubated with or without doxycycline to induce SF-1 and then treated with angiotensin II (Ang II). Aldosterone was measured by immunoassay. SF-1 mRNA was silenced by small interfering RNA (siRNA) by Nucleofector technology. mRNA levels were measured by real-time RT-PCR. Ang II treatment without doxycycline increased aldosterone production by 11.3-fold and CYP11B2 mRNA by 116-fold. Doxycycline treatment increased SF-1 mRNA levels by 3.7-fold and inhibited Ang II-induced aldosterone by 84%. Doxycycline treatment inhibited Ang II-stimulated CYP11B2 mRNA levels by 86%. Doxycycline decreased basal CYP11B2 promoter activity by 68%. Doxycycline inhibited Ang II stimulation by 85%. Ang II increased CYP21 mRNA expression by 4.6-fold, whereas doxycycline inhibited induction by 69%. In contrast, doxycycline treatment increased CYP11B1 mRNA by 1.7-fold in basal cells and increased Ang II induction by 3.6-fold. SF-1-specific siRNA significantly reduced SF-1 mRNA expression as compared with cells treated with control siRNA. SF-1 siRNA reversed doxycycline stimulation of CYP B1 and its inhibition of CYP11B2. However, in H295R/TR/SF-1 cells without doxycycline treatment, both CYP11B1 and CYP11B2 mRNAs were significantly decreased, suggesting that both enzymes require a minimal level of SF-1 for basal expression. In summary, SF-1 overexpression dramatically inhibited CYP11B2 expression and decreased aldosterone production. The opposing effects of SF-1 on CYP11B1 and CYP11B2 suggest that the regulation of SF-1 activity may play a role that determines the relative ability to produce mineralocorticoid and glucocorticoid.

Figure 1

Doxycycline (Doxy) regulation of SF-1 mRNA level in the H295R/TR/SF-1 cell line. SF-1 mRNA expression was normalized by basal SF-1 mRNA levels in the H295R/TR/SF-1 cell line with and without Ang II (10 nm) or doxycycline (0.25 μg/ml) treatment. The cells were treated with or without doxycycline for 72 h and then with or without Ang II for 24 h. Real-time quantitative RT-PCR was used to measure SF-1 mRNA expression. Values represent the mean ± se from three independent experiments each run in triplicate. *, P < 0.05; **, P < 0.01.

Figure 2

Effects of elevated SF-1 on aldosterone and cortisol production. H295R/TR/SF-1 cells were incubated with doxycycline (Doxy; 0.25 μg/ml) for 72 h. Cells were then treated with or without Ang II (10 nm) for 24 h. Aldosterone (A) and cortisol (B) levels were then measured in the experimental medium. Values represent the mean ± se from three independent experiments each ran in triplicate. *, P < 0.01.

Figure 3

SF-1 effects on steroidogenic enzyme mRNA expression. H295R/TR/SF-1 cells were treated with doxycycline (Doxy; 0.25 μg/ml) for 72 h followed by treatment with or without Ang II (10 nm) for 24 h. Real-time quantitative RT-PCR was used to measure CYP11B1 (A), CYP11B2 (A), CYP21 (A), CYP11A1 (B), HSD3B2 (B), and StAR (B) mRNA expression. Values represent the mean ± se from three independent experiments each run in triplicate. *, P < 0.05; **, P < 0.01.

Figure 4

Effects of elevated SF-1 on CYP11B1 or CYP11B2 promoter activity H295R/TR/SF-1 cells were incubated with doxycycline (Doxy; 0.25 μg/ml) for 72 h. The cells were then transfected with luciferase reporter vectors containing the 5′-flanking region of CYP11B1 and CYP11B2 gene. After recovery, cells were treated with Ang II (10 nm) for 6 h followed by measurement of luciferase reporter activity. Values represent the mean ± se from three independent experiments each run in triplicate. *, P < 0.05; **, P < 0.01.

Figure 5

Effects of SF-1 siRNA on SF-1, CYP11B1, and CYP11B2 expression. Both endogenous and transgene SF-1 expression were silenced in H295R/TR/SF-1 cells by RNA interference using Nucleofector technology. Cells were transfected with 10 nm SF-1-specific Stealth siRNA or with a control siRNA oligo (scrambled oligo) and allowed to recover for 48 h. During the final 24 h, cells were treated with or without doxycycline (0.25 μg/ml) and then harvested for total RNA isolation and real-time quantitative RT-PCR. Data points represent the mean ± se from three independent experiment. *, P < 0.01.