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Impaired steroidogenic factor 1 (NR5A1) activity in mutant Y1 mouse adrenocortical tumor cells
C Frigeri1, J Tsao, W Czerwinski
1Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Abstract:
Mutants isolated from the Y1 mouse adrenocortical tumor cell line (clones 10r-9 and 10r-6) are resistant to ACTH because they fail to express the melanocortin-2 receptor (MC2R). In this study, we show that a luciferase reporter plasmid driven by 1,800 bp of the proximal promoter region of the MC2R was expressed poorly in the mutant cells compared with parent Y1 cells. The differential expression of the MC2R in parent and mutant cells resulted from impaired activity of the orphan nuclear receptor NR5A1 (SF1) on the promoter as determined by 5'-deletion analysis. Furthermore, the activity of an SF1 expression plasmid on an SF1-dependent reporter plasmid was compromised in mutant clones. The site-specific DNA binding properties of SF1 from parent and mutant cells did not differ as determined in electrophoretic mobility shift assays, and the addition of the activation domain of VP16 to the amino terminus of SF1 restored the transcriptional activity of the protein. In addition, the levels of SF1 and other cofactors including WT1, CBP/p300, and steroid receptor coactivator 1 did not differ appreciably between parent and mutant cells. Taken together, these results suggest that ACTH resistance in the mutant clones resulted from a defect that affected the activation properties of SF1 rather than its DNA binding activity. Consistent with the observed impairment in SF1 function, other SF1-dependent genes, including Cyp11b1 and steroidogenic acute regulatory protein (StAR), were poorly expressed 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 requirement for SF1. These mutants thus provide an experimental paradigm to identify factors that influence SF1 function and to evaluate the relative importance of SF1 in the expression of genes essential for adrenal steroidogenesis.
Insights
ACTH resistance in Y1 cells stems from impaired activator function of the nuclear receptor SF1, not its DNA binding. This defect impacts steroidogenesis gene expression and overall steroid production.
Area of Science:
- Endocrinology
- Molecular Biology
- Genetics
Background:
- Adrenocorticotropic hormone (ACTH) resistance in Y1 mouse adrenocortical tumor cells (clones 10r-9 and 10r-6) is linked to the absence of the melanocortin-2 receptor (MC2R).
- The orphan nuclear receptor NR5A1 (SF1) is crucial for regulating genes involved in steroidogenesis.
Purpose of the Study:
- To investigate the molecular basis of ACTH resistance in Y1 cell mutants.
- To elucidate the role of SF1 activity in MC2R gene expression and adrenal steroidogenesis.
Main Methods:
- Luciferase reporter assays to assess MC2R promoter activity.
- 5'-deletion analysis to map SF1-responsive regions.
- Electrophoretic mobility shift assays (EMSAs) to evaluate SF1 DNA binding.
- Western blotting to determine protein levels of SF1 and cofactors.
- Steroid production assays using 22(R)-hydroxycholesterol.
Main Results:
- MC2R promoter activity was significantly reduced in mutant Y1 cells compared to parent cells.
- Impaired SF1 activity, not DNA binding, was identified as the cause of reduced MC2R expression.
- Restoration of SF1 transcriptional activity was achieved by adding the VP16 activation domain.
- Expression of other SF1-dependent genes (Cyp11b1, StAR) and overall steroidogenesis were also impaired in mutant cells.
Conclusions:
- ACTH resistance in these Y1 mutants is due to a defect in SF1's activation function.
- These mutants serve as a model to study factors influencing SF1 function and its role in adrenal steroidogenesis.
- Differential gene expression suggests varying SF1 dependencies for MC2R, Cyp11a, Cyp11b1, and StAR.