Role of Sp1 in cAMP-dependent transcriptional regulation of the bovine CYP11A gene

The pituitary peptide hormone ACTH regulates transcription of the cholester ol side chain cleavage cytochrome P450 (CYP11A) gene via cAMP and activatio n of cAMP-dependent protein kinase. A G-rich sequence element conferring cA MP-dependent regulation has been found to reside within region -118 to -100 of the bovine CYP11A promoter. Previous studies have suggested that it bin ds a protein antigenically related to the transcription factor Sp1. We now report that the -118/-100 element binds both Sp1 and Sp3, members of the Sp family of transcription factors. me have made use of Drosophila SL2 cells, which lack endogenous Sp factors, to dissect the possible functional roles of Sp1, Sp3, and Sp4. All factors stimulated the activity of cotransfected reporter constructs in which the promoter of the bovine CYP11A gene regula tes luciferase expression. Sp3 did not repress Sp1-dependent activation, as has previously been shown for other G-rich promoters. Mutation of the -118 /-100 element of CYP11A abolished Sp1-mediated activation of a CYP11A repor ter gene in SL2 cells as well as cAMP responsiveness in human H295R cells. Furthermore, cotransfection of SL2 cells with the catalytic subunit of cAMP -dependent protein kinase together with Sp1 and a CYP11A reporter construct enhanced Sp1-dependent activation of the reporter 4.2-fold, demonstrating that Sp1 confers cAMP responsiveness in these cells. Thus, we show that int roduction of Sp1 alone in an Sp-negative cell such as SL2 is sufficient to achieve the cAMP-dependent regulation observed using the -118/ -100 element of CYP11A in adrenocortical cells.