Androstenedione Is the Preferred Substrate for Cytochrome P450 11β-hydroxylase Leading to the Production of 11β-Hydroxyandrostenedione in the Adrenal Gland

Abstract

Abstract Adrenal cytochrome P450 11β-hydroxylase (CYP11B1) is a mitochondrial enzyme that catalyzes the final step of glucocorticoid synthesis, converting 11-deoxycortisol (S) and deoxycorticosterone (DOC) to cortisol (F) and corticosterone (CORT), respectively. CYP11B1 is predominantly located in the zona fasciculata of the adrenal gland with studies also showing that CYP11B1 catalyzes the conversion of androstenedione (A4) and testosterone (T) to 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT), respectively. Adrenal vein sampling in women has shown that 11OHA4 turnover is high, with a marked increase after treatment with ACTH which is known to upregulate CYP11B1 expression. We hypothesized that CYP11B1’s affinity for A4 as a substrate may be favored over glucocorticoids since 11OHA4 is one of the major androgens produced in the adrenalThis study aimed to elucidate the kinetic parameters (Km and Vmax) for A4 and T with respect to CYP11B1. A4 and T (0.2 µM to 5 µM) were assayed in HEK-293 cells transiently transfected with CYP11B1 and the adrenodoxin redox partner. Data was used to generate progress curves and fitted to the Michaelis-Menten equation. A4 had the lowest Km (0.21 μM) with a significantly higher Vmax (315.77 pmol/min/mg protein) in comparison to T, S and DOC. Results suggest that A4 binds more readily to CYP11B1 resulting in the high turnover of substrate. The androgenic activity of CYP11B1 catalyzed steroid products was determined using a luciferase assay conducted in CV1 cells. Both A4 and 11OHA4 showed no androgenic activity, however, the 11βHSD2 product of 11OHA4, 11-ketoandrostenedione (11KA4), elicited a response at 100 nM. 11OHT was an androgen receptor (AR) agonist, however, exhibiting a lower response when compared to T and 11-ketotestosterone over all concentrations tested (1, 10 and 100 nM). As confirmation of CYP11B1 activity in the adrenal, circulatory steroid levels were determined in healthy female subjects (n=88). CORT, F and 11OHA4 were measured at a frequency of 70–100%, compared to 11OHT (40–70%), while 11βHSD2 activity produced 11KA4 (<10%). In conclusion, the catalytic efficiency of CYP11B1 towards A4 is higher compared to T and the classical substrates. The high substrate affinity and turnover provides evidence for 11OHA4 in adrenal vein samples. Concurrently, our analysis suggests that agonism is diminished by the presence of the C11- hydroxyl group, while AR agonistic activity is gained upon its conversion to a keto group. Furthermore, these androgens could perhaps modulate cortisol production in the adrenal due to potential competition between precursor substrates.