Inhibition of hormone-stimulated steroidogenesis in cultured Leydig tumor cells by a cholesterol-linked phosphorothioate oligodeoxynucleotide antisense to diazepam-binding inhibitor.
The polypeptide diazepam-binding inhibitor (DBI) has been previously shown to stimulate testicular Leydig, adrenocortical, and glial-cell mitochondrial steroidogenesis in vitro. To assess the in situ role of DBI in trophic hormone-stimulated steroidogenesis, we suppressed DBI levels in the hormone-responsive MA-10 Leydig tumor cells, using a cholesterol-linked phosphorothioate oligodeoxynucleotide (Chol-odN) antisense to DBI. Treating MA-10 cells with Chol-odN antisense to DBI resulted in a dose-dependent reduction of DBI levels (ED50 = 1 microM). In contrast, Chol-odN sense to DBI did not affect its expression. Saturating amounts of human choriogonadotropin (hCG) increased MA-10 progesterone production by 150-fold. Addition of increased concentrations of Chol-odNs sense to DBI or of a nonrelated sequence did not reduce the MA-10 response to hCG. However, in the presence of Chol-odN antisense to DBI that could reduce DBI levels, MA-10 cells lost their ability to respond to hCG (ED50 = 1 microM). In these studies the hCG-stimulated cAMP levels and cytochrome P450 side-chain cleavage activity, as measured by metabolism of 22(R)-hydroxycholesterol, were not affected by the Chol-odNs used. These observations provide unequivocal evidence that DBI plays a vital role in the acute stimulation of steroidogenesis by trophic hormones.
Department of Anatomy and Cell Biology, Georgetown University Medical Center, Washington, DC 20007.
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Boujrad
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Hudson
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Papadopoulos
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D002784:Cholesterol / Q000494:pharmacology*; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D026261:Diazepam Binding Inhibitor; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology; D007700:Kinetics; D007984:Leydig Cell Tumor; D051379:Mice; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011374:Progesterone / Q000096:biosynthesis*; D013256:Steroids / Q000096:biosynthesis*; D013873:Thionucleotides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D006063:Chorionic Gonadotropin; D026261:Diazepam Binding Inhibitor; D006888:Hydroxycholesterols; D016376:Oligonucleotides, Antisense; D013256:Steroids; D013873:Thionucleotides; C003585:22-hydroxycholesterol; D011374:Progesterone; D002784:Cholesterol; D000242:Cyclic AMP