Costs and Consequences of Cellular Compartmentalization and Substrate Competition among Human Enzymes Involved in Androgen and Estrogen Synthesis

Abstract

The impact of compartmental expression of steroidogenic enzymes and of changes in flux through delta5 and delta4 metabolism on sex steroid synthesis was investigated by rebuilding pathways using recombinant enzyme expression by infection of insect cells with recombinant baculovirus constructs. Human cytochromes 17alpha-hydroxylase/17,20-lyase (P450c17) and aromatase (P450arom), always coexpressed with their redox partner NADPH-P450 oxidoreductase (CPR) and 3beta-hydroxysteroid dehydrogenase/delta5-4 isomerase (3betaHSD; types 1 or 2), were compartmentally expressed in different cell populations or coexpressed together with pregnenolone (100 nM) as substrate. Estrone was compared among cell compartments expressing different enzyme combinations or in cells coexpressing all enzymes (experiment 1). Additionally, P450c17, 3betaHSD, and CPR were all coexpressed, and androstenedione was measured in cells with different 3betaHSD expression levels or activity using an inhibitor, trilostane (experiment 2). Steroids were measured by immunoassay and mass spectrometry. In experiment 1, partitioning of P450c17, P450arom, and 3betaHSD markedly decreased estrone synthesis in comparison to cells coexpressing enzymes in different combinations. However, partitioning P450arom with 3betaHSD from P450c17 in different cell populations resulted in more estrone than either of the other two-cell compartment models. In experiment 2 (cells coexpressing P450c17, 3betaHSD, and CPR), androstenedione secretion was (paradoxically) higher at lower levels of 3betaHSD, and partial inhibition of 3betaHSD by trilostane also increased androstenedione when 3betaHSD expression was high. We conclude 1) that tissue or cell-specific, partitioned expression of sex steroid synthesizing enzymes limits rather than maximizes estrogen synthesis and 2) that limiting metabolism by 3betaHSD can paradoxically promote androgen synthesis when 3betaHSD expression is high by promoting delta5-steroid flux.

Equine 5α-reductase activity and expression in epididymis

“…Substrate infusion studies conducted in vivo are complicated by multiple tissue compartments and competing enzymes, most notably 3β-HSD, where pregnenolone is concerned in studies using placenta (Chavatte et al 1995). Even in the simplest of cell systems, compartmental expression of enzymes can markedly alter the secreted steroid products in unexpected counterintuitive ways (Conley et al 2011). Directly testing this hypothesis in other less complex experiments is difficult for many reasons, not least of which is the lack of any purified preparation of mammalian 5α-reductase.…”

Steroidogenic enzyme activities in the pre- and post-parturient equine placenta

“…Reactions for both 5α-reductase and P450c17 activities were supported by co-incubation with NADPH and a generating system (1 mM NADP+, 10 mM glucose-6-phosphate and 1.25 U glucose-6-phosphate dehydrogenase). The activity of 3βHSD was examined essentially as described earlier (Conley et al 2011), with minor modifications, from the accumulation of androstenedione from the added substrate, dehydroepiandrosterone (DHEA; 3 μM). Incubations were conducted in the presence of 1 mM βNAD.…”

The Synthesis and Metabolism of Steroid Hormones

“…7 This cooperation is exemplified by estradiol synthesis in the ovarian follicle, where luteinizing hormone (LH) acts on the theca cells to stimulate production of androgen precursors and follicle-stimulating hormone (FSH) acts on granulosa cells to stimulate aromatization of these androgens into estrogens. Indeed, the requirement for cooperative efforts by two different tissues or cell types is a characteristic of estrogen biosynthesis.…”