THE ORPHAN NUCLEAR RECEPTOR STEROIDOGENIC FACTOR-I REGULATES THE CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-MEDIATED TRANSCRIPTIONAL ACTIVATION OF RAT CYTOCHROME P450C17 (17-ALPHA-HYDROXYLASE C17-20 LYASE)/

The rat steroid cytochrome P450 17 alpha-hydroxylase/c17-20 lyase (rP4 50c17) gene is transcriptionally regulated in steroidogenic tissues. P revious studies showed that one DNA element located between -75 and -5 0 base pairs (bp) upstream from the transcriptional initiation site me diated both the basal and cAMP-regulated transcription of rP450c17. Us ing a series of mutant oligonucleotides in gel mobility shift assays a nd in functional assays, it is now shown that a core sequence of 12 bp , located at -58/-69 bp, is essential for nuclear protein binding and transcriptional activation. Mutant oligonucleotides cloned into a luci ferase reporter gene construct containing a heterologous thymidine kin ase promoter, transfected into mouse Leydig MA-10 and adrenocortical Y -1 cells, gave results consistent with those of gel shift assays. Muta nts that abolished binding of the nuclear protein to DNA abolished the basal transcription of the gene as well as the responsiveness to cAMP , whereas those mutants that did not abolish binding of the nuclear pr otein to DNA still showed strong basal transcription as well as respon siveness to cAMP. Comparison of the binding sequence with the consensu s binding site for the orphan nuclear receptor steroidogenic factor-1 (SF-1) showed that eight of nine bases were identical. However, the se quence from rP450c17 includes an additional three bases at the 5'-end, not previously demonstrated to be important for SF-1 binding. Recombi nant rat SF-1 protein expressed in Escherichia coil binds to this sequ ence, and antibodies raised against rat SF-1 abolish binding of both r ecombinant SF-1 and the nuclear protein from Y-1 and MA-10 cells. Thes e observations demonstrate that this region of the rP450c17 gene is re sponsible for both the basal transcription and cAMP inducibility and i s bound by the orphan nuclear receptor SF-1. It is further shown that SF-1 can be phosphorylated in vitro by protein kinase A. This phosphor ylation occurs at serine and threonine residues and results in decreas ed binding to the rP450c17 -58/-69 element. Since SF-1 mediates cAMP-i nduced transcriptional regulation of the rat P450c17 gene, phosphoryla tion of SF-1 via protein kinase A is likely to play a regulatory role in transcriptional activation.