Frigeri C, Tsao J, Czerwinski W, Schimmer B P
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Mol Endocrinol. 2000 Apr;14(4):535-44. doi: 10.1210/mend.14.4.0440.
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.
从Y1小鼠肾上腺皮质肿瘤细胞系分离出的突变体（克隆10r - 9和10r - 6）对促肾上腺皮质激素（ACTH）具有抗性，因为它们无法表达促黑素细胞激素-2受体（MC2R）。在本研究中，我们发现与亲代Y1细胞相比，由MC2R近端启动子区域的1800 bp驱动的荧光素酶报告质粒在突变细胞中的表达较差。通过5' - 缺失分析确定，亲代细胞和突变细胞中MC2R的差异表达是由于孤儿核受体NR5A1（SF1）对启动子的活性受损所致。此外，在突变克隆中，SF1表达质粒对SF1依赖性报告质粒的活性受到损害。电泳迁移率变动分析结果表明，亲代细胞和突变细胞中SF1的位点特异性DNA结合特性没有差异，并且将VP16的激活结构域添加到SF1的氨基末端可恢复该蛋白的转录活性。此外，亲代细胞和突变细胞之间SF1以及其他辅助因子（包括WT1、CBP/p300和类固醇受体辅激活因子1）的水平没有明显差异。综上所述，这些结果表明，突变克隆中的ACTH抗性是由影响SF1激活特性而非其DNA结合活性的缺陷所致。与观察到的SF1功能受损一致，其他SF1依赖性基因，包括Cyp11b1和类固醇生成急性调节蛋白（StAR），表达较差，并且22(R)-羟基胆固醇向类固醇产物的代谢所证明的整体类固醇生成受损。有趣的是，MC2R、Cyp11a、Cyp11b1和StAR转录本受到的影响程度不同，这表明这些基因中的每一个对SF1可能有不同的绝对需求。因此，这些突变体提供了一个实验范例，用于鉴定影响SF1功能的因素，并评估SF1在肾上腺类固醇生成所必需的基因表达中的相对重要性。
