Costs and consequences of cellular compartmentalization and substrate competition among human enzymes involved in androgen and estrogen synthesis

Alan J Conley, Cynthia J. Corbin, James L. Thomas, Nancy A. Gee, Bill L. Lasley, Benjamin Moeller, Scott D Stanley, Trish Berger

Research output: Contribution to journalArticle

16 Citations (Scopus)

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.

Original languageEnglish (US)
Article number15
JournalBiology of Reproduction
Volume86
Issue number1
DOIs
StatePublished - Jan 2012

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Androgens
Estrogens
Costs and Cost Analysis
Enzymes
Aromatase
Androstenedione
Estrone
Cardiopulmonary Resuscitation
Steroids
Steroid 17-alpha-Hydroxylase
Pregnenolone
Isomerases
Hydroxysteroid Dehydrogenases
Baculoviridae
Cytochromes
Mixed Function Oxygenases
NADP
Immunoassay
Population
Oxidation-Reduction

Keywords

  • Androgen
  • Compartmental expression
  • Estradiol
  • Estrogen
  • Follicle
  • Ovary
  • P450
  • Placenta
  • Reproduction
  • Sex steroid synthesis
  • Steroid hormones
  • Steroidogenesis
  • Two-cell model

ASJC Scopus subject areas

  • Cell Biology

Cite this

Costs and consequences of cellular compartmentalization and substrate competition among human enzymes involved in androgen and estrogen synthesis. / Conley, Alan J; Corbin, Cynthia J.; Thomas, James L.; Gee, Nancy A.; Lasley, Bill L.; Moeller, Benjamin; Stanley, Scott D; Berger, Trish.

In: Biology of Reproduction, Vol. 86, No. 1, 15, 01.2012.

Research output: Contribution to journalArticle

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AU - Moeller, Benjamin

AU - Stanley, Scott D

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AB - 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.

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KW - Follicle

KW - Ovary

KW - P450

KW - Placenta

KW - Reproduction

KW - Sex steroid synthesis

KW - Steroid hormones

KW - Steroidogenesis

KW - Two-cell model

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