Impaired steroidogenic factor 1 (NR5A1) activity in mutant Y1 mouse adrenocortical tumor cells

Mutants isolated from the Y1 mouse adrenocortical tumor cell line (clones 1 0r-9 and 10r-6) are resistant to ACTH because they fail to express the mela nocortin-2 receptor (MC2R). In this study, we show that a luciferase report er plasmid driven by 1800 bp of the proximal promoter region of the MC2R wa s expressed poorly in the mutant cells compared with parent Y1 cells. The d ifferential expression of the MC2R in parent and mutant cells resulted from impaired activity of the orphan nuclear receptor NR5A1 (SF1) on the promot er as determined by 5'-deletion analysis. Furthermore, the activity of an S F1 expression plasmid on an SF1-dependent reporter plasmid was compromised in mutant clones. The site-specific DNA binding properties of SF1 from pare nt and mutant cells did not differ as determined in electrophoretic mobilit y shift assays, and the addition of the activation domain of VP16 to the am ino terminus of SF1 restored the transcriptional activity of the protein. I n addition, the levels of SF1 and other cofactors including WT1, CBP/p300, and steroid receptor coactivator 1 did not differ appreciably between paren t and mutant cells. Taken together, these results suggest that ACTH resista nce in the mutant clones resulted from a defect that affected the activatio n properties of SF1 rather than its DNA binding activity. Consistent with t he observed impairment in SF1 function, other SF1-dependent genes, includin g Cyp11b1 and steroidogenic acute regulatory protein (StAR), were poorly ex pressed and global steroidogenesis, as evidenced by the metabolism of 22(R) -hydroxycholesterol to steroid products, was impaired. Interestingly, MC2R, Cyp11a, Cyp11b1, and StAR transcripts were not affected to the same degree , suggesting that each of these genes may have a different absolute require ment for SF1. These mutants thus provide an experimental paradigm to identi fy factors that influence SF1 function and to evaluate the relative importa nce of SF1 in the expression of genes essential for adrenal steroidogenesis .