| First Author | Legeza B | Year | 2013 |
| Journal | Endocrinology | Volume | 154 |
| Issue | 1 | Pages | 205-13 |
| PubMed ID | 23183177 | Mgi Jnum | J:354168 |
| Mgi Id | MGI:7731047 | Doi | 10.1210/en.2012-1778 |
| Citation | Legeza B, et al. (2013) The microsomal enzyme 17beta-hydroxysteroid dehydrogenase 3 faces the cytoplasm and uses NADPH generated by glucose-6-phosphate dehydrogenase. Endocrinology 154(1):205-13 |
| abstractText | Recent studies proposed a functional coupling between 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3)-dependent testosterone formation and 11beta-hydroxysteroid dehydrogenase 1 (11beta-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 17beta-HSD3 and/or 11beta-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. 17beta-HSD3-dependent reduction of Delta4-androstene-3,17-dione was affected by neither coexpression with 11beta-HSD1 nor overexpression or knockdown of hexose-6-phosphate dehydrogenase. In contrast, knockdown of glucose-6-phosphate dehydrogenase decreased 17beta-HSD3 activity, indicating dependence on cytoplasmic NADPH. Upon selective permeabilization of the plasma membrane by digitonin, 17beta-HSD3 but not 11beta-HSD1 was detected by antibodies against C-terminal epitope tags, suggesting a cytoplasmic orientation of 17beta-HSD3. The cytoplasmic orientation was confirmed using proteinase K digestion of microsomal preparations and by analysis of glycosylation of wild-type 17beta-HSD3 and chimera in which the N-terminal anchor sequences between 17beta-HSD3 and 11beta-HSD1 were exchanged. In conclusion, the results demonstrate a cytoplasmic orientation of 17beta-HSD3 and dependence on glucose-6-phosphate dehydrogenase-generated NADPH, explaining the lack of a direct functional coupling with the luminal 11beta-HSD1-mediated glucocorticoid metabolism. |
