Abstract
Androgens are the obligatory precursors of estrogens. In humans, classic androgen biosynthesis yields testosterone, thought to represent the predominant circulating active androgen both in men and women. However, recent work has shown that 11-ketotestosterone, derived from the newly described 11-oxygenated androgen biosynthesis pathway, makes a substantial contribution to the active androgen pool in women. Considering that classic androgens are the obligatory substrates for estrogen biosynthesis catalyzed by cytochrome P450 aromatase, we hypothesized that 11-oxygenated androgens are aromatizable. Here we use steroid analysis by tandem mass spectrometry to demonstrate that human aromatase generates 11-oxygenated estrogens from 11-oxygenated androgens in 3 different cell-based aromatase expression systems and in human ex vivo placenta explant cultures. We also show that 11-oxygenated estrogens are generated as a byproduct of the aromatization of classic androgens. We show that 11β-hydroxy-17β-estradiol binds and activates estrogen receptors α and β and that 11β-hydroxy-17β-estradiol and the classic androgen pathway-derived active estrogen, 17β-estradiol, are equipotent in stimulating breast cancer cell line proliferation and expression of estrogen-responsive genes. 11-oxygenated estrogens were, however, not detectable in serum from individuals with high aromatase levels (pregnant women) and elevated 11-oxygenated androgen levels (patients with congenital adrenal hyperplasia or adrenocortical carcinoma). Our data show that while 11-oxygenated androgens are aromatizable in vitro and ex vivo, the resulting 11-oxygenated estrogens are not detectable in circulation, suggesting that 11-oxygenated androgens function primarily as androgens in vivo.
Highlights
In humans, androstenedione (A4) is the immediate substrate for testosterone biosynthesis
We show that 11β-hydroxy-17β-estradiol binds and activates estrogen receptors α and β and that 11β-hydroxy-17β-estradiol and the classic androgen pathway-derived active estrogen, 17β-estradiol, are equipotent in stimulating breast cancer cell line proliferation and expression of estrogen-responsive genes. 11-oxygenated estrogens were, not detectable in serum from individuals with high aromatase levels and elevated 11-oxygenated androgen levels
Despite being overlooked for several decades, it is clear that 11-oxygenated androgens make a substantial contribution to the circulating androgen pool, in women [1,2,3,4,5]
Summary
HEK293, COS-1, and JEG3 cells were purchased from the American Type Culture Collective. Aromatase activity in the MCF7, MCF7arom, and JEG3 cells were compared using a tritiated water assay as previously reported [12]. The MCF7arom and JEG3 cells were treated with 1 μM of the appropriate steroid (A4, T, 11OHA4, 11OHT, 11KA4, 11KT with and without 10-μM letrozole) for 24 hours. At the start of the assay, 50-μL starved medium was added to each well of an E-plate 16 (ACEA Biosciences) and left to equilibrate for 30 minutes before taking a background reading During this time a suspension of the MCF7-BUS cells (100 000 cells/mL) was prepared in starvation medium. BrdU-based cell proliferation assay MCF7arom cells were starved with phenol red-free and serum-free medium for 24 hours before treatment with the indicated androgen and drug treatments.
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