TRANSCRIPTIONAL REGULATION OF RAT CYTOCHROME P450C17 EXPRESSION IN MOUSE LEYDIG MA-10 AND ADRENAL Y-1 CELLS - IDENTIFICATION OF A SINGLE PROTEIN THAT MEDIATES BOTH BASAL AND CAMP-INDUCED ACTIVITIES

Cytochrome P450c17, 17 alpha-hydroxylase/17,20 lyase, is a key enzyme in the steroidogenic pathway leading to the production of corticostero ids and androgens from the adrenal gland and sex steroids from the gon ads. Both enzymatic activities of the protein are encoded by a single gene, CYP1 7, which is expressed in both the human adrenal and gonad b ut not in the placenta, and in the rodent gonad and placenta but not t he rodent adrenal. We isolated and sequenced a full-length rat genomic clone (7,553 bases) containing the entire coding region of the rat P4 50c17 gene, and all intronic sequences and 1,560 bp of 5'-flanking DNA (EMBL Acc#X69816). To determine which sequences in the rat P450c17 pr omoter may be responsible for basal and cAMP-stimulated gene transcrip tion, deletion constructs containing between -1,560 and -53 base pairs of 5'-flanking DNA from the rat P450c17 gene were ligated to plasmids expressing the reporter gene luciferase and transfected into two mous e cell lines, adrenal Y-1 cells, and testicular Leydig MA-10 cells. Hi ghest basal and cAMP-stimulated luciferase activity were found in cons tructions containing 156 bp of 5'-flanking DNA. This construction cont ains a sequence very similar to the consensus cis element reported to be responsible for cAMP enhancement of the rat somatostatin gene and a lso overlaps a sequence similar to the consensus element for the orpha n steroid receptor SF-1. Gel mobility-shift analysis, using a 30-bp ol igonucleotide containing this region incubated with cellular extracts from cultured mouse adrenal Y-1 and mouse Leydig MA-10 cells, revealed all the extracts to contain two proteins that bind to this sequence. Neither DNA-protein complex was further retarded by co-incubation with an anti-CREB antibody, suggesting that cAMP regulation of this gene o ccurs via a non-CREB protein. Mutation of this oligonucleotide resulte d in loss of binding of only one of these proteins, but resulted in lo ss of both basal and cAMP stimulation of rat P450c17 promoter-regulate d gene transcription. Southwestern analysis suggests that one of these proteins is larger than SF-1. This study suggests that a protein that binds to an SF-1 like sequence regulates both basal and cAMP-stimulat ed rat P450c17 gene expression in rodent cells.