During studies on the regulation of rat ovarian steroidogenic enzymes by interleukin-1 (IL-1), we observed substantial metabolism of 25-hydroxycholesterol to two unusual polar products. This unexpected effect was observed both in isolated granulosa cells and in whole ovarian dispersates and was also induced by tumor necrosis factor , but not by insulin-like growth factor I or follicle-stimulating hormone. The effect was dependent on time and the dose of IL-1 and was blocked by an IL-1 receptor antagonist. The formation of the polar metabolites was inhibited by ketoconazole and trilostane, but not by aminoglutethimide. Subsequent purification of these novel metabolites and analysis by gas chromatography/mass spectrometry, NMR, and high performance liquid chromatography revealed them to be related 7-hydroxylated hydroxycholesterols (cholest-4-ene-7,25-diol-3-one and cholest-5-ene-3,7,25-triol). IL-1-stimulated ovarian 7-hydroxylase activity (3-10 pmol/min/mg of cellular protein) was nearly 4-fold that of control levels using 25-hydroxycholesterol as substrate. Activities at or below control levels were observed when IL-1-treated cell sonicates were boiled or assayed in the presence of NADH (rather than NADPH), indicating that involvement of a nonenzymatic process was unlikely. IL-1-stimulated 7-hydroxylase activity was inhibited to basal levels by a 10-fold excess of unlabeled 25- or 27-hydroxycholesterol, but not by cholesterol, pregnenolone, progesterone, testosterone, or dehydroepiandrosterone, suggesting that ovarian 7-hydroxylase is specific for hydroxycholesterols. Furthermore, when IL-1-treated ovarian cultures were incubated with radiolabeled cholesterol or testosterone, no 7-hydroxylated products were observed. We were also unable to detect any mRNA transcripts for liver cholesterol 7-hydroxylase in IL-1-stimulated ovarian cultures. This study describes an ovarian hydroxycholesterol 7-hydroxylase that differs from liver cholesterol 7-hydroxylase and from other nonhepatic progestin/androgen 7-hydroxylases. The novel finding of the regulation of a 7-hydroxylase by IL-1 (and tumor necrosis factor ) suggests a unique role for cytokines in the regulation of cholesterol metabolism in the ovary and possibly other tissues.

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