This Article Full Text Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Martens, J. W. M. Articles by Miller, W. L. Search for Related Content PubMed PubMed Citation Articles by Martens, J. W. M. Articles by Miller, W. L.

Enzymatic Activities of P450c17 Stably Expressed in Fibroblasts from Patients with the Polycystic Ovary Syndrome¹

Departments of Pediatrics (J.W.M.M., D.H.G., W.A., R.J.A., H.R., W.L.M.) and Pathology (V.S.O.), University of California, San Francisco, California 94143; and Department of Medicine, Brigham and Women’s Hospital (A.D.), Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Prof. Walter L. Miller, Department of Pediatrics, Building MR-IV, Room 209, University of California, San Francisco, California 94143-0978.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting approximately 5–10% of women of reproductive age. The clinical features of PCOS include oligo/anovulation, hyperandrogenemia, and hyperinsulinemia. Because P450c17 is the single enzyme catalyzing both 17-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 enzyme’s 17,20-lyase activity, thereby favoring androgen production, and that serine phosphorylation of the insulin receptor ß-chain (IR-ß) inhibits IR-ß tyrosine phosphorylation, causing insulin resistance in vitro. We previously suggested that a gain of function mutation in a single serine kinase might cause the hyperandrogenism and insulin resistance observed in PCOS patients by excessive phosphorylation of both P450c17 and IR-ß. To test this hypothesis, we obtained fibroblasts from nine previously studied patients: three controls, three PCOS patients with normal levels of IR-ß serine phosphorylation, and three PCOS patients with increased levels of IR-ß serine phosphorylation. Initial studies showed that such skin fibroblasts could not be transfected effectively by calcium phosphate, diethylaminoethyl-dextran, lipofection or adenovirus procedures. Therefore, we employed a retroviral infection system to stably express human P450c17 in the primary cultures of fibroblast cells from the PCOS patients and controls and measured the resulting 17-hydroxylase and 17,20-lyase activity. The cells were analyzed in a blinded fashion until the study was complete. The 17-hydroxylase and 17,20-lyase activities in each cell line correlated well with the amount of P450c17 protein expressed, but there was no correlation between either enzymatic activity (or their ratio) with the clinical phenotype of the cells’ donors even when results were corrected for the number of P450c17 complementary DNA inserts per cell line. Overnight incubation with 1 µmol/L insulin also did not affect enzymatic activity. Thus, we were unable to find evidence for the hypothesis that in PCOS a single abnormal kinase hyperphosphorylates both IR-ß, causing insulin resistance, and P450c17, causing hyperandrogenism. However, because fibroblasts do not normally express either P450c17 or the accessory proteins needed for its optimal activity, these results cannot exclude a role for serine phosphorylation in the hyperandrogenism and insulin resistance of PCOS.

This article has been cited by other articles:

				H. F. Escobar-Morreale, M. Luque-Ramirez, and J. L. San Millan The Molecular-Genetic Basis of Functional Hyperandrogenism and the Polycystic Ovary Syndrome Endocr. Rev., April 1, 2005; 26(2): 251 - 282. [Abstract] [Full Text] [PDF]

				N. Huang and W. L. Miller LBP Proteins Modulate SF1-Independent Expression of P450scc in Human Placental JEG-3 Cells Mol. Endocrinol., February 1, 2005; 19(2): 409 - 420. [Abstract] [Full Text] [PDF]

				W.-H. Yang, L. B. Lutz, and S. R. Hammes Xenopus laevis Ovarian CYP17 Is a Highly Potent Enzyme Expressed Exclusively in Oocytes. EVIDENCE THAT OOCYTES PLAY A CRITICAL ROLE IN XENOPUS OVARIAN ANDROGEN PRODUCTION J. Biol. Chem., March 7, 2003; 278(11): 9552 - 9559. [Abstract] [Full Text] [PDF]

				M. Li, J. F. Youngren, A. Dunaif, I. D. Goldfine, B. A. Maddux, B. B. Zhang, and J. L. Evans Decreased Insulin Receptor (IR) Autophosphorylation in Fibroblasts from Patients with PCOS: Effects of Serine Kinase Inhibitors and IR Activators J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4088 - 4093. [Abstract] [Full Text] [PDF]

				V. L. Nelson, K.-n. Qin, R. L. Rosenfield, J. R. Wood, T. M. Penning, R. S. Legro, J. F. Strauss III, and J. M. McAllister The Biochemical Basis for Increased Testosterone Production in Theca Cells Propagated from Patients with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., December 1, 2001; 86(12): 5925 - 5933. [Abstract] [Full Text] [PDF]