Enzymatic activities of p450c17 stably expressed in fibroblasts from patients with the polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting a pproximately 5-10% of women of reproductive age. The clinical features of P COS include oligo/anovulation, hyperandrogenemia, and hyperinsulinemia. Bec ause P450c17 is the single enzyme catalyzing both 17 alpha -hydroxylase and 17,20-lyase activities in the ovary and adrenal, some have suggested that defects in P450c17 may cause the hyperandrogenism of PCOS. Previous studies have shown that serine hyperphosphorylation of P450c17 increases the enzym e's 17,20-lyase activity, thereby favoring androgen production, and that se rine phosphorylation of the insulin receptor beta -chain (IR-beta) inhibits IR-beta tyrosine phosphorylation, causing insulin resistance in vitro. We previously suggested that a gain of function mutation in a single serine ki nase might cause the hyperandrogenism and insulin resistance observed in PC OS patients by excessive phosphorylation of both P450c17 and IR-beta. To te st this hypothesis, we obtained fibroblasts from nine previously studied pa tients: three controls, three PCOS patients with normal levels of IR-beta s erine phosphorylation, and three PCOS patients with increased levels of IR- beta serine phosphorylation. Initial studies showed that such skin fibrobla sts could not be transfected effectively by calcium phosphate, diethylamino ethyl-dextran, lipofection or adenovirus procedures. Therefore, we employed a retroviral infection system to stably express human P450c17 in the prima ry cultures of fibroblast cells from the PCOS patients and controls and mea sured the resulting 17 alpha -hydroxylase and 17,20-lyase activity. The cel ls were analyzed in a blinded fashion until the study was complete. The 17 alpha -hydroxylase and 17,20-lyase activities in each cell line correlated well with the amount of P450c17 protein expressed, but there was no correla tion between either enzymatic activity (or their ratio) with the clinical p henotype of the cells' donors even when results were corrected for the numb er of P450c17 complementary DNA inserts per cell line. Overnight incubation with 1 mu mol/L insulin also did not affect enzymatic activity. Thus, we w ere unable to find evidence for the hypothesis that in PCOS a single abnorm al kinase hyperphosphorylates both IR-beta, causing insulin resistance, and P450c17, causing hyperandrogenism. However, because fibroblasts do not nor mally express either P450c17 or the accessory proteins needed for its optim al activity, these results cannot exclude a role for serine phosphorylation in the hyperandrogenism and insulin resistance of PCOS.