Assessment of the ability of type 2 cytochrome b5 to modulate 17,20-lyase activity of human P450c17

Abstract

The 17α-hydroxylase and 17,20-lyase activities of P450c17 lead to the production of 17α-hydroxypregnenolone (17α-OH-Preg) and dehydroepiandrosterone (DHEA), respectively, in different tissues. The mechanisms of differential regulation of these two activities are not yet fully elucidated. It has been previously shown that cytochrome b5 (cyt- b5) could facilitate the 17,20-lyase activity of human P450c17. Recently, a cDNA (type 2 cyt- b5) sharing 45.8% homology with type 1 cyt- b5 has been isolated from human testis. Since high 17,20-lyase activity is required for the production of androgens in the testis, we wanted to determine the importance of this second cDNA in the modulation of P450c17 17,20-lyase activity and hence, its role in the formation of active androgens. We therefore isolated type 2 cyt- b5 from human testis by RT-PCR and analyzed, by transient transfection in transformed human embryonic kidney cells (HEK-293) of various amounts of vectors expressing cyt- b5, P450-reductase and P450c17, its ability to modulate the 17,20-lyase activity of human P450c17. Results show that, in the presence of NADPH cytochrome P450 reductase (P450-red), type 2 cyt- b5 increases 17,20-lyase activity to a level comparable to that of type 1. These results support the idea that types 1 and 2 cyt- b5 could be involved in the differential modulation of 17α-hydroxylase and 17,20-lyase activities of P450c17. Furthermore, the analysis of mRNA expression of types 1 and 2 cyt- b5 by RT-PCR using primers specific to each type showed that both types are present in the liver but also in the adrenal and testis.