Regulation of P450 oxidoreductase by gonadotropins in rat ovary and its effect on estrogen production

Abstract

Background: P450 oxidoreductase (POR) catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS), and about half the patients with ABS have ambiguous genitalia and/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs) differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we examined the regulation of POR expression in ovarian granulosa cells by gonadotropins, and its possible role in steroidogenesis.

Methods: Changes in gene expression in MSCs during differentiation into steroidogenic cells were examined by DNA microarray analysis. Changes in mRNA and protein expression of POR in the rat ovary or in granulosa cells induced by gonadotropin treatment were examined by reverse transcription-polymerase chain reaction and western blotting. Effects of transient expression of wild-type or mutant (R457H or V492E) POR proteins on the production of estrone in COS-7 cells were examined in vitro. Effects of POR knockdown were also examined in estrogen producing cell-line, KGN cells.

Results: POR mRNA was induced in MSCs following transduction with the SF-1 retrovirus, and was further increased by cAMP treatment. Expression of POR mRNA, as well as Cyp19 mRNA, in the rat ovary were induced by equine chorionic gonadotropin and human chorionic gonadotropin. POR mRNA and protein were also induced by follicle stimulating hormone in primary cultured rat granulosa cells, and the induction pattern was similar to that for aromatase. Transient expression of POR in COS-7 cells, which expressed a constant amount of aromatase protein, greatly increased the rate of conversion of androstenedione to estrone, in a dose-dependent manner. The expression of mutant POR proteins (R457H or V492E), such as those found in ABS patients, had much less effect on aromatase activity than expression of wild-type POR proteins. Knockdown of endogenous POR protein in KGN human granulosa cells led to reduced estrone production, indicating that endogenous POR affected aromatase activity.

Conclusion: We demonstrated that the expression of POR, together with that of aromatase, was regulated by gonadotropins, and that its induction could up-regulate aromatase activity in the ovary, resulting in a coordinated increase in estrogen production.

Figure 1

Induction of POR mRNA in human MSCs. A: RT-PCR analysis of POR mRNA in UE7T-13 cells transduced with SF-1 or pQCXIP (control) virus and cultured with or without 8-Br-cAMP for 2 d. B: Results of real-time PCR are also shown. Data are the mean ± SEM values of duplicate experiments.

Figure 2

Time-dependent changes in POR and aromatase mRNA expression in immature rat ovaries treated with eCG and hCG. A: Expression of each mRNA was examined by RT-PCR. Immature rat ovaries were primed with 30 IU eCG for 48 h, followed with treatment with 30 IU hCG for the indicated times. B: Results of real-time PCR are also shown. Data are the mean ± SEM values of at least three independent experiments. *, P < 0.01.

Figure 3

Regulation of POR and aromatase mRNA and protein levels by FSH. Granulosa cells isolated from immature DES-primed rats were cultured and treated with FSH (30 ng/ml) for the indicated times, and the total RNA or protein was extracted. A: RT-PCR was performed with the specific primer sets for POR, aromatase, and GAPDH. B: Results of real-time PCR are also shown. Data are the mean ± SEM values of at least three independent experiments. *, P < 0.01. C: Western blot analysis was performed with antibodies to POR, aromatase, and GAPDH, using the same lysates.

Figure 4

Production of estrone by co-transfection of wild-type or mutant POR and aromatase expression vectors in COS-7 cells. A and B: Amounts of estrone in the culture medium were detected by EIA at the indicated times and for the indicated contents of POR expression vector, with or without aromatase expression vector. The protein expression of POR, aromatase, and GAPDH after transfection with these amounts of vectors was detected by western blot analysis. C and D: COS-7 cells were transfected with wild-type POR or R457H or V492E mutant expression vectors (20 ng/well) with aromatase expression vector, at the indicated times. Data are means and SEM values (B) or SD values (D) of at least three assays, respectively.

Figure 5

Effect of POR knockdown on estrone production and cAMP response of POR and aromatase genes in KGN cells. After transfection with the indicated siRNAs, the culture medium was replaced with that containing 1 mM 8-Br-cAMP and 10 μM androstenedione, and the amount of estrone in the culture medium was detected by EIA 48 h later (B and C). Data are means and SEM values of at least three assays. *, Significant difference in estrone production between KGN cells and KGN cells transfected siRNA for POR#1 or #2 (P < 0.01). The protein expression under these culture conditions at the indicated times is also shown (A and B).