Characterization of the adrenal cytochrome P450C17 in the hamster, a small animal model for the study of adrenal dehydroepiandrosterone biosynthesis.

Abstract

The hamster, like the human produces cortisol as its major glucocorticoid, rather than corticosterone, typical of most enzyme rodents. It is not known, however, if the hamster cytochrome P450C17 (P450C17), a key enzyme for cortisol formation, also exhibits 17,20-lyase activity and if it catalyzes the formation of dehydroepiandrosterone (DHEA) at the adrenal level. To study this, we isolated the cDNA of P450C17 from a hamster adrenal library. This cDNA was sequenced and was found to have an open reading frame for a protein of 511 amino acids, as compared to the human P450C17, which contains 508 amino acids. The hamster P450C17 cDNA, in the coding region, is 76% homologous with the human P450C17 cDNA. The cDNA was then cloned in the expression vector pSV-SPORT 1, which was transiently transfected into COS 1 cells. The transfected cells were used for temporal studies on the transformation of radiolabeled C21-delta5- and C21-delta4-precursors. When transfected cells were incubated with [14C]pregnenolone, rapid formation of [14C]DHEA occurred. The intermediate 17alpha-hydroxypregnenolone accumulated initially with subsequent metabolism to DHEA. Likewise, when incubated with C21-delta4-steroids, [14C]progesterone and [3H]17alpha-hydroxyprogesterone, the 17,20-lyase product androstenedione was produced efficiently. In these studies, with respect to the delta5 pathway, the expressed hamster P450C17 gave similar results to bovine P450C17 cDNA inserted in the same expression vector. However, in contrast to the bovine enzyme, which converted low amounts of progesterone to androstenedione, the expressed hamster P450C17 enzyme showed an active metabolism via the delta4 pathway. Northern blot analysis, using the complete alpha-32P labeled hamster P450C17 cDNA as the probe, demonstrated a strong presence of P450C17 mRNA in hamster adrenals, a weaker presence in testes and ovaries, and no detectable species in brain, mesentery, and kidney. Immunoblotting analysis using an anti-rat P450C17 antibody demonstrated the presence of P450C17 protein in hamster adrenals, testes, and ovaries. Hamster adrenal cell suspensions and microsomal preparations were used to demonstrate the biosynthesis of [14C]17alpha-hydroxypregnenolone and [14C]DHEA from [14C]pregnenolone; both metabolites were formed during incubations. However, the ratio of [14C]DHEA/[14C]17alpha-hydroxypregnenolone was much lower in adrenal cells than in transfected COS 1 cells, indicating the presence of putative factors in hamster adrenal cells, favoring the 17alpha-hydroxylase activity rather than that of the 17,20-lyase. In conclusion, these studies demonstrate that the hamster adrenal is both a DHEA and a cortisol producer, and, therefore, this animal could be a suitable small animal model for the study of the role of DHEA in relation to human biochemistry and physiology.