A NOVEL NONHEPATIC HYDROXYCHOLESTEROL 7-ALPHA-HYDROXYLASE THAT IS MARKEDLY STIMULATED BY INTERLEUKIN-1-BETA - CHARACTERIZATION IN THE IMMATURE RAT OVARY

During studies on the regulation of rat ovarian steroidogenic enzymes by interleukin-1 beta (IL-1 beta), we observed substantial metabolism of 25-hydroxycholesterol to two unusual polar products. This unexpecte d effect was observed both in isolated granulosa cells and in whole ov arian dispersates and was also induced by tumor necrosis factor alpha but not by insulin-like growth factor I or follicle-stimulating hormon e. The effect was dependent on time and the dose of IL-1 beta and was blocked by an IL-1 receptor antagonist, The formation of the polar met abolites was inhibited by ketoconazole and trilostane, but not by amin oglutethimide. Subsequent purification of these novel metabolites and analysis by gas chromatography/mass spectrometry, NMR, and high perfor mance liquid chromatography revealed them to be related 7 alpha-hydrox ylated hydroxycholesterols (cholest-4-ene-7 alpha,25-diol-3-one and ch olest-5-ene-3 beta,7 alpha 25-triol). IL-1 beta-stimulated ovarian 7 a lpha-hydroxylase activity (3-10 pmol/min/mg of cellular protein) was n early 4-fold that of control levels using 25-hydroxycholesterol as sub strate, Activities at or below control levels were observed when IL-1 beta-treated cell sonicates were boiled or assayed in the presence of NADH (rather than NADPH), indicating that involvement of a nonenzymati c process was unlikely, IL-1 beta-stimulated 7 alpha-hydroxylase activ ity was inhibited to basal levels by a 10-fold excess of unlabeled 25- or 27-hydroxycholesterol, but not by cholesterol, pregnenolone, proge sterone, testosterone, or dehydroepiandrosterone, suggesting that ovar ian 7 alpha-hydroxylase is specific for hydroxycholesterols, Furthermo re, when IL-1 beta-treated ovarian cultures were incubated with radiol abeled cholesterol or testosterone, no 7 alpha-hydroxylated products w ere observed, We were also unable to detect any mRNA transcripts for l iver cholesterol 7 alpha-hydroxylase in IL-1 beta-stimulated ovarian c ultures, This study describes an ovarian hydroxycholesterol 7 alpha-hy droxylase that differs from liver cholesterol 7 alpha-hydroxylase and from other nonhepatic progestin/androgen 7 alpha-hydroxylases, The nov el finding of the regulation of a 7 alpha-hydroxylase by IL-1 beta (an d tumor necrosis factor alpha) suggests a unique role for cytokines in the regulation of cholesterol metabolism in the ovary and possibly ot her tissues.