h-89 and 22-hydroxycholesterol

It has been suggested that the signal transduction pathway initiated by apoA-I activates key proteins involved in cellular lipid efflux. We investigated apoA-I-mediated cAMP signaling in cultured human fibroblasts induced with (22R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Treatment of stimulated fibroblasts with apoA-I for short periods of time (

Topics: Alitretinoin; Alleles; Animals; Apolipoprotein A-I; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Biotinylation; Catalytic Domain; Cell Membrane; CHO Cells; Colforsin; Cricetinae; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; Humans; Hydroxycholesterols; Isoquinolines; Lipid Metabolism; Mutation; Phosphorylation; Precipitin Tests; Protein Binding; Signal Transduction; Sulfonamides; Tangier Disease; Time Factors; Transfection; Tretinoin

We demonstrate the mechanism by which Cordyceps sinensis (CS) mycelium regulates Leydig cell steroidogenesis. Mouse Leydig cells were treated with forskolin, H89, phorbol 12-myristate 13-acetate, staurosporine, or steroidogenic enzyme precursors with or without 3 mg/ml CS; then testosterone production was determined. H89, but not phorbol 12-myristate 13-acetate or staurosporine, decreased CS-treated Leydig cell steroidogenesis. CS inhibited Leydig cell steroidogenesis by suppressing the activity of P450scc enzyme, but not 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase, 20alpha-hydroxylase, or 17beta-hydroxysteroid dehydrogenase enzymes. Thus, CS activated the cAMP-protein kinase A signal pathway, but not protein kinase C, and attenuated P45scc enzyme activity to reduce human chorionic gonadotropin-stimulated steroidogenesis in purified mouse Leydig cells.

Topics: Androstenedione; Animals; Cells, Cultured; Colforsin; Cordyceps; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Hydroxycholesterols; Isoquinolines; Leydig Cells; Male; Mice; Mice, Inbred C57BL; Mycelium; Progestins; Protein Kinase C; Staurosporine; Sulfonamides; Testosterone; Tetradecanoylphorbol Acetate

RLD-1 and OR-1 are closely related orphan nuclear receptors that can be activated by certain oxysterols. To obtain cells stably expressing RLD-1 or OR-1, CHOK1 cells were successively transfected with a DGRE2-ALP reporter and GR-RLD-1 or GR-OR-1 chimeric constructs. The selected cell clones that showed low background activity of the reporter and maximum fold induction by 22R(OH)cholesterol were used for subsequent experiments. Treatment of the cells with PGE2, TPA, or 8-bromo-cAMP alone did not transactivate the reporter. However, the induction of the reporter by 22R(OH)cholesterol was markedly enhanced in the presence of PGE2, TPA, 8-bromo-cAMP, or forskolin in cells expressing GR-RLD-1. The enhancement was inhibited by H-89 and bisindolylmaleimide, both inhibitors of protein kinases. These results suggest that transactivation by ligand-activated RLD-1 may be further modulated/regulated through other signal transduction pathways involving phosphorylation catalyzed by protein kinases.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; CHO Cells; Colforsin; Cricetinae; Cyclic AMP-Dependent Protein Kinases; Dinoprostone; DNA-Binding Proteins; Drug Synergism; Enzyme Inhibitors; Genes, Reporter; Hydroxycholesterols; Indoles; Isoquinolines; Liver X Receptors; Maleimides; Orphan Nuclear Receptors; Protein Kinase C; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Receptors, Thyroid Hormone; Signal Transduction; Sulfonamides; Tetradecanoylphorbol Acetate; Transfection