title: The microsomal enzyme 17β-hydroxysteroid dehydrogenase 3 faces the cytoplasm and uses NADPH generated by glucose-6-phosphate dehydrogenase.
abstract: Recent studies proposed a functional coupling between 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3)-dependent testosterone formation and 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1)-mediated interconversion of glucocorticoids through competition for the luminal pyridine nucleotide pool. To test this hypothesis, we used human embryonic kidney-293 cells transfected with 17β-HSD3 and/or 11β-HSD1, in the absence or presence of hexose-6-phosphate dehydrogenase that generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the endoplasmic reticulum and determined enzyme activities. As an endogenous cell model, mouse MA-10 Leydig cells were used. 17β-HSD3-dependent reduction of Δ4-androstene-3,17-dione was affected by neither coexpression with 11β-HSD1 nor overexpression or knockdown of hexose-6-phosphate dehydrogenase. In contrast, knockdown of glucose-6-phosphate dehydrogenase decreased 17β-HSD3 activity, indicating dependence on cytoplasmic NADPH. Upon selective permeabilization of the plasma membrane by digitonin, 17β-HSD3 but not 11β-HSD1 was detected by antibodies against C-terminal epitope tags, suggesting a cytoplasmic orientation of 17β-HSD3. The cytoplasmic orientation was confirmed using proteinase K digestion of microsomal preparations and by analysis of glycosylation of wild-type 17β-HSD3 and chimera in which the N-terminal anchor sequences between 17β-HSD3 and 11β-HSD1 were exchanged. In conclusion, the results demonstrate a cytoplasmic orientation of 17β-HSD3 and dependence on glucose-6-phosphate dehydrogenase-generated NADPH, explaining the lack of a direct functional coupling with the luminal 11β-HSD1-mediated glucocorticoid metabolism.
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
authors.forename: Balázs
authors.initials: B
authors.lastname: Legeza
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Balázs
authors.forename: Lyubomir G
authors.initials: LG
authors.lastname: Nashev
authors.forename: Alex
authors.initials: A
authors.lastname: Odermatt
mesh_terms: D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1 / Q000378:metabolism; D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000818:Animals; D002237:Carbohydrate Dehydrogenases / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008861:Microsomes / Q000201:enzymology*; D008954:Models, Biological; D009249:NADP / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D009249:NADP; D015067:17-Hydroxysteroid Dehydrogenases; C476565:17beta-hydroxysteroid dehydrogenase type 3; D002237:Carbohydrate Dehydrogenases; C020552:galactose-6-phosphate dehydrogenase; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1