The microsomal enzyme 17β-hydroxysteroid dehydrogenase 3 faces the cytoplasm and uses NADPH generated by glucose-6-phosphate dehydrogenase

Endocrinology. 2013 Jan;154(1):205-13. doi: 10.1210/en.2012-1778. Epub 2012 Nov 26.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 11-beta-Hydroxysteroid Dehydrogenase Type 1 / metabolism
  • 17-Hydroxysteroid Dehydrogenases / metabolism*
  • Animals
  • Carbohydrate Dehydrogenases / metabolism*
  • Cell Line
  • Cytoplasm / metabolism*
  • Humans
  • Male
  • Mice
  • Microsomes / enzymology*
  • Models, Biological
  • NADP / metabolism*
  • Protein Binding

Substances

  • NADP
  • 17-Hydroxysteroid Dehydrogenases
  • 17beta-hydroxysteroid dehydrogenase type 3
  • Carbohydrate Dehydrogenases
  • galactose-6-phosphate dehydrogenase
  • 11-beta-Hydroxysteroid Dehydrogenase Type 1