An investigation into the metabolism of classic and 11-oxygenated androgens in peripheral blood mononuclear cells

In both sexes, androgens are responsible for the initiation and maintenance of sexual differentiation and reproduction, as well as the development of secondary male characteristics including male pattern body hair, muscle bulk, sexual function and reproductive capacity. Androgens are also key regulators of immune cell function and have a suppressive influence on differentiation, proliferation, and cytokine production. Local generation of active androgens from circulating androgen precursors is an essential mediator of androgen action in peripheral androgen target cells and tissues. To examine the activation of classic and 11-oxygenated androgens within human peripheral blood mononuclear cells (PBMCs), PBMCs were isolated from healthy donors and subsequent activity analysis assays were carried out. PBMCs were incubated ex vivo with androgen precursors and active androgens from the classic pathway (DHEA, A4 and T) and the 11-oxygenated androgen pathway (11KA4, 11KT, 11OHA4 and 11OHT). In total, 12 steroids were quantified in 50% (v/v) methanol in water by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The expression of genes encoding steroidogenic enzymes was assessed by quantitative PCR (qPCR). qPCR showed that the enzyme AKR1C3, rather than HSD17B3, was the major reductive 17β-hydroxysteroid dehydrogenase in PBMCs, responsible for the activation of the 11-oxygenated androgen 11-ketotestosterone and the classic androgen testosterone. Approximately 8-fold more 11-ketotestosterone than testosterone was produced from their respective precursors, indicating preferential activation of 11-oxygenated androgens by AKR1C3. These results identified 11-ketotestosterone as the major active androgen in PBMCs. RNAseq analysis of FACS-sorted PBMC subpopulations (dice-database.org) revealed natural killer cells to be the major location of AKR1C3 activity and expression. The enzyme SRD5A1 catalysed the 5α-reduction of classic, but not 11-oxygenated androgens in PBMCs. Overall, this project has revealed that 11-oxygenated androgens are the favoured substrate for AKR1C3 in PBMCs, predominantly due to natural killer cell AKR1C3 activity, producing the major active androgen 11-ketotestosterone