A novel nonhepatic hydroxycholesterol 7α-hydroxylase that is markedly stimulated by interleukin-1β. Characterization in the immature rat ovary

During studies on the regulation of rat ovarian steroidogenic enzymes by interleukin-1β (IL-1β), 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 α, but not by insulin-like growth factor I or follicle-stimulating hormone. The effect was dependent on time and the dose of IL-1β 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 7α- hydroxylated hydroxycholesterols (cholest-4-ene-7α,25-diol-3-one and cholest-5-ene-3β,7α,25-triol). IL-1β-stimulated ovarian 7α-hydroxylase activity (3-10 pmol/mini/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-1β-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-1β-stimulated 7α-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 7α-hydroxylase is specific for hydroxycholesterols. Furthermore, when IL-1β-treated ovarian cultures were incubated with radiolabeled cholesterol or testosterone, no 7α-hydroxylated products were observed. We were also unable to detect any mRNA transcripts for liver cholesterol 7α- hydroxylase in IL-1β-stimulated ovarian cultures. This study describes an ovarian hydroxycholesterol 7α-hydroxylase that differs from liver cholesterol 7α-hydroxylase and from other nonhepatic progestin/androgen 7α- hydroxylases. The novel finding of the regulation of a 7α-hydroxylase by IL- 1β (and tumor necrosis factor α) suggests a unique role for cytokines in the regulation of cholesterol metabolism in the ovary and possibly other tissues.