title: Basal transcriptional activity and cyclic adenosine 3',5'-monophosphate responsiveness of the human cytochrome P450scc promoter transfected into MA-10 Leydig cells.
abstract: Mouse Leydig MA-10 tumor cells are a good model of testicular steroidogenesis. The endogenous murine P450scc mRNA in these cells accumulated in response to 8-bromo-cAMP, forskolin, cholera toxin, and 1-methyl-3-isobutylxanthine, but not in response to 1,9-dideoxyforskolin, indicating that this accumulation was stimulated by the protein kinase-A pathway. Inhibiting transcription with actinomycin-D showed that the half-life of cytochrome P450scc mRNA in these cells was not altered by cAMP, consistent with earlier nuclear run-on data showing that the effect of cAMP on P450scc is at the transcriptional level. A series of 17 fragments of 5'-flanking DNA from the human P450scc gene were fused to the gene for firefly luciferase and transiently transfected into MA-10 cells. The longest construct, containing 2327 basepairs of 5'-flanking DNA, responded 4-fold to forskolin and, hence, was used to optimize the forskolin dose response, showing that 30 microM forskolin elicited a 90% maximal effect. Examination of the activity of the deletion constructs located basal and cAMP-responsive sequences. Constructions containing 79 basepairs of 5'-flanking DNA had basal activity; adding sequences between -79 and -110 had minimal effect, but adding sequences between -110 and -127 increased basal activity 3-fold. Adding sequences beyond -127 did not increase basal transcription further, indicating the presence of a basal transcription element between -110 and -127. These serial deletion mutants were used similarly to locate cAMP responsiveness between -1620 and -1676, indicating the presence of a cAMP response element in this region. The locations of these basal and cAMP-responsive sequences correspond well with those previously identified when human P450scc promoter/reporter constructions were transfected into mouse adrenocortical Y-1 cells, but differ from those identified when such constructions were transfected into human JEG-3 choriocarcinoma cells.
authors.affiliation: Department of Pediatrics, University of California, San Francisco 94143-0978.
authors.forename: D W
authors.initials: DW
authors.lastname: Hum
authors.forename: B
authors.initials: B
authors.lastname: Staels
authors.forename: S M
authors.initials: SM
authors.lastname: Black
authors.forename: W L
authors.initials: WL
authors.lastname: Miller
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics* / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D007985:Leydig Cells; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D003609:Dactinomycin; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D008156:Luciferases; D002786:Cholesterol Side-Chain Cleavage Enzyme; D015056:1-Methyl-3-isobutylxanthine