SUN-361 Cultured Murine Osteoblasts Convert DHEA to Testosterone

Abstract

Androgens have complex effects on the skeleton. Besides gonadal testosterone (T), the adrenal produces the androgen precursor dehydroepiandrosterone (DHEA), using 3β- and 17β-hydroxysteroid dehydrogenases (3βHSD & 17βHSD) to convert to T in target tissues. Microarray analyses of murine calvarial osteoblasts, RT-PCR of long bone osteoblasts, and RNA-Seq of human bone biopsies confirmed that each of the enzyme families is expressed in osteoblasts, suggesting osteoblasts can generate androgens from the adrenal-derived androgen precursor DHEA. Activation of osteoblast androgen receptor (AR) signaling by DHEA was detected using an AR reporter construct, providing evidence that active androgens are generated. To understand how DHEA is converted to T, we treated murine primary osteoblasts with 100 nM and 1 μM DHEA, or vehicle control. Conditioned media were collected 1, 2, and 3 days after DHEA treatment and assayed for intermediate and active androgens by tandem mass spectrometry with two-dimensional chromatography. As DHEA was consumed, the androgen intermediates androstenediol (A5) and androstenedione (A4) were generated and subsequently converted to T. The peak concentrations of T generated by DHEA 100 nM and 1 μM were 22 and 101 pg/ml, respectively. The equilibrium dissociation constant of the AR for T is ~0.2 nM (57.7 pg/ml), indicating sufficient T production to activate AR in androgen-sensitive osteoblasts. Cultured osteoblasts preferentially converted DHEA to A5, via 17βHSD, rather than to A4, signifying that in the conversion of DHEA to T, 3βHSD is the rate-limiting step. Of the 13 17βHSD isoforms, 7 were expressed in these samples. In contrast, only a single gene isoform of Hsd3b—Hsd3b7—was abundantly expressed in mouse osteoblasts and human bone. We investigated the effects of 3βHSD7 in osteoblasts. An Hsd3b7 shRNA knocked-down mRNA and protein expression by >85%, and caused an osteoblast growth defect compared to an shRNA control. 3βHSD7 has known functions in bile acid synthesis, converting 7α-hydroxycholesterol to 7α-hydroxycholestenone (7HC). Treatment of Hsd3b7 knockdown osteoblasts with 7HC rescued the growth defect suggesting that osteoblasts might generate 7HC or a subsequent metabolite as a trophic factor. We now report an unreported function of bone as a source of T, by conversion of the adrenal androgen precursor DHEA, using 3βHSD7 as a common enzyme for androgen and bile acid synthesis, and leading to the activation of osteoblast AR signaling. These data suggest that the skeleton has evolved protective mechanisms against hypogonadal bone loss that exploits the continued production of adrenal DHEA. This concept is especially important in men with prostate cancer bone metastasis undergoing testicular-targeted therapies whereby adrenal DHEA may continue to fuel cancer growth, and in bone maturation during adrenarche before the pubertal rise in gonadal androgens.