Targeting human glutathione transferase A3-3 attenuates progesterone production in human steroidogenic cells

Abstract

hGSTA3-3 (human Alpha-class glutathione transferase 3-3) efficiently catalyses steroid Δ5–Δ4 double-bond isomerization in vitro, using glutathione as a cofactor. This chemical transformation is an obligatory reaction in the biosynthesis of steroid hormones and follows the oxidation of 3β-hydroxysteroids catalysed by 3β-HSD (3β-hydroxysteroid dehydrogenase). The isomerization has commonly been ascribed to a supplementary function of 3β-HSD. The present study is the first to provide evidence that hGSTA3-3 contributes to this step in steroid hormone biosynthesis in complex cellular systems. First, we find glutathione-dependent Δ5–Δ4 isomerase activity in whole-cell extracts prepared from human steroidogenic cells. Secondly, effective inhibitors of hGSTA3-3 dramatically decrease the conversion of Δ5-androstene-3,17-dione into Δ4-androstene-3,17-dione in cell lysates. Thirdly, we show that RNAi (RNA interference) targeting hGSTA3-3 expression decreases by 30% the forskolin-stimulated production of the steroid hormone progesterone in a human placental cell line. This effect is achieved at low concentrations of two small interfering RNAs directed against distinct regions of hGSTA3-3 mRNA, and is weaker in unstimulated cells, in which hGSTA3-3 expression is low. The results concordantly show that hGSTA3-3 makes a significant contribution to the double-bond isomerization necessary for steroid hormone biosynthesis and thereby complements the indispensable 3β-hydroxysteroid oxidoreductase activity of 3β-HSD. The results indicate that the lower isomerase activity of 3β-HSD is insufficient for maximal rate of cellular sex hormone production and identify hGSTA3-3 as a possible target for pharmaceutical intervention in steroid hormone-dependent diseases.