Differential effects of high and low steroidogenic factor-1 expression on CYP11B2 expression and aldosterone production in adrenocortical cells.
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.
Ye
Ping
P
Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912, USA.
Nakamura
Yashuhiro
Y
Lalli
Enzo
E
Rainey
William E
WE
D000302:Adrenal Cortex / Q000187:drug effects / Q000378:metabolism*; D000450:Aldosterone / Q000378:metabolism*; D000804:Angiotensin II / Q000494:pharmacology; D002478:Cells, Cultured; D019405:Cytochrome P-450 CYP11B2 / Q000235:genetics* / Q000378:metabolism; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D005938:Glucocorticoids / Q000378:metabolism; D006801:Humans; D008901:Mineralocorticoids / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics / Q000378:metabolism; D054339:Steroidogenic Factor 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
D005938:Glucocorticoids; D008901:Mineralocorticoids; D034741:RNA, Small Interfering; D054339:Steroidogenic Factor 1; D000804:Angiotensin II; D000450:Aldosterone; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase; D004318:Doxycycline