Basal transcriptional activity and cyclic adenosine 3′, 5′-monophosphate responsiveness of the human cytochrome P450scc promoter transfected into MA-10 leydig cells

Dean W. Hum, Bart Staels, Stephen Matthew Black, Walter L. Miller

Research output: Contribution to journalArticle

40 Citations (Scopus)

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 l-methyl-3-isobutylxanthine, but not in response to 1, 9-dideoxy-forskolin, 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 µM forskolin elicited a 90% maximal effect. Exami nation 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-l cells, but differ from those identified when such constructions were transfected into human JEG-3 choriocarcinoma cells.

Original languageEnglish (US)
Pages (from-to)546-552
Number of pages7
JournalEndocrinology
Volume132
Issue number2
DOIs
StatePublished - Jan 1 1993

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Cholesterol Side-Chain Cleavage Enzyme
Leydig Cells
Adenosine
Colforsin
DNA
Firefly Luciferases
8-Bromo Cyclic Adenosine Monophosphate
Messenger RNA
Choriocarcinoma
Cholera Toxin
Response Elements
Dactinomycin
Cyclic AMP-Dependent Protein Kinases
Genes
Half-Life

ASJC Scopus subject areas

  • Endocrinology

Cite this

Basal transcriptional activity and cyclic adenosine 3′, 5′-monophosphate responsiveness of the human cytochrome P450scc promoter transfected into MA-10 leydig cells. / Hum, Dean W.; Staels, Bart; Black, Stephen Matthew; Miller, Walter L.

In: Endocrinology, Vol. 132, No. 2, 01.01.1993, p. 546-552.

Research output: Contribution to journalArticle

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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 l-methyl-3-isobutylxanthine, but not in response to 1, 9-dideoxy-forskolin, 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 µM forskolin elicited a 90{\%} maximal effect. Exami nation 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-l cells, but differ from those identified when such constructions were transfected into human JEG-3 choriocarcinoma cells.",
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