Conversion of pregnenolone to DHEA by human 17alpha-hydroxylase/17, 20-lyase (P450c17). Evidence that DHEA is produced from the released intermediate, 17alpha-hydroxypregnenolone.
Most previous studies using reconstituted systems and fast kinetics suggest that the conversion of pregnenolone to dehydroepiandrosterone (DHEA; the precursor of androgen and estrogen biosynthesis) by P450c17 does not require the release of the intermediate 17alpha-OHPreg (a precursor of cortisol biosynthesis). With such a mechanism, it is difficult to conceive how high amounts of DHEA may be produced in some cells or tissues, such as the testis and cells from the adrenal reticularis, while in other tissues such as the fasciculata zone, high levels of 17alpha-OHPreg are synthesized. In this report, we address this matter using intact transfected cells, which better reflect the actual cellular conditions. Furthermore, by using transfected cells, we can conveniently analyze human enzymes, as we are not restricted by the availability of human tissues as in the case of methods using purified or partially purified enzymes. Using intact HEK-293 cells transfected with human P450c17 in culture, we showed, in a time course study of the transformation of pregnenolone, that there is an accumulation of 17alpha-OHPreg, and that, subsequently, the accumulated 17alpha-OHPreg decreases with a concomitant increase in DHEA production. The DHEA/17alpha-OHPreg ratio changes from 0.1 :1 after 1 h incubation to 50 : 1 after 20 h. This result strongly suggests that the transformation of Preg to DHEA proceeds through two steps in which DHEA is produced from the released intermediate 17alpha-OHPreg. We also show that high levels of substrate vs. enzyme concentration will lead to high hydroxylase activity whereas the reverse will increase the lyase activity. The result is in good agreement with recent observations suggesting that surrounding enzymes and steroids could modulate the lyase activity. Cotransfection of vectors expressing cytochrome b5 and NADPH cytochrome P450 reductase indicates that both are required for an optimum production of DHEA.
Soucy
P
P
Medical Research Council Group in Molecular Endocrinology, Oncology and Molecular Endocrinology Research Center, CHUQ pavillon CHUL and Laval University, Ste-Foy, Quebec, Canada.
Luu-The
V
V
D006907:17-alpha-Hydroxypregnenolone / Q000378:metabolism*; D002460:Cell Line; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism; D003687:Dehydroepiandrosterone / Q000096:biosynthesis*; D006801:Humans; D007668:Kidney; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D011284:Pregnenolone / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D011993:Recombinant Fusion Proteins; D006907:17-alpha-Hydroxypregnenolone; D003687:Dehydroepiandrosterone; D011284:Pregnenolone; D015786:Cytochromes b5; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase