Crawford P A, Sadovsky Y, Milbrandt J
Department of Pathology and Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Mol Cell Biol. 1997 Jul;17(7):3997-4006. doi: 10.1128/MCB.17.7.3997.
The orphan nuclear receptor steroidogenic factor 1 (SF-1) is expressed in the adrenal gland and gonads and is an important regulator of the expression of cytochrome P-450 steroidogenic enzymes in cultured cells. Targeted disruption of the SF-1 gene in mice shows that it is a critical participant in the genetic program that promotes the development of urogenital mesoderm into the adrenal gland and gonads. To assess the ability of SF-1 to regulate this differentiation pathway, we ectopically expressed SF-1 in murine embryonic stem (ES) cells. We found that stable expression of SF-1 is sufficient to alter ES cell morphology, permit cyclic AMP (cAMP) and retinoic acid-induced expression of the endogenous side chain cleavage enzyme gene, and consequently, promote steroidogenesis. While steroid production is dependent upon SF-1, cAMP induction of steroidogenesis does not enhance the responsiveness of an SF-1-specific reporter. Furthermore, the activity of a P450SCC promoter/luciferase reporter construct, which is induced by cAMP in steroidogenic cells and ES cells converted by stable expression of SF-1, is not induced by cAMP in wild-type ES cells transiently transfected with SF-1, suggesting that the induction of downstream gene products is required before steroidogenesis can occur. We demonstrate that mutants which disrupt the DNA binding domain or the AF2 transcriptional activation domain of SF-1 do not confer the steroidogenic phenotype to ES cells. Interestingly, however, AF2 mutants fused to the VP16 activation domain do confer the steroidogenic phenotype to ES cells, but only in the presence of a portion of the ligand binding domain. These studies extend the role of SF-1 in steroidogenic tissues to that of a dominant regulator of the steroidogenic cell phenotype.
孤儿核受体类固醇生成因子1（SF-1）在肾上腺和性腺中表达，是培养细胞中细胞色素P-450类固醇生成酶表达的重要调节因子。在小鼠中靶向破坏SF-1基因表明，它是促进泌尿生殖中胚层发育为肾上腺和性腺的遗传程序中的关键参与者。为了评估SF-1调节这种分化途径的能力，我们在小鼠胚胎干细胞（ES细胞）中异位表达了SF-1。我们发现，SF-1的稳定表达足以改变ES细胞形态，允许环磷酸腺苷（cAMP）和视黄酸诱导内源性侧链裂解酶基因的表达，从而促进类固醇生成。虽然类固醇的产生依赖于SF-1，但cAMP对类固醇生成的诱导并没有增强SF-1特异性报告基因的反应性。此外，P450SCC启动子/荧光素酶报告基因构建体的活性，在类固醇生成细胞和通过SF-1稳定表达转化的ES细胞中由cAMP诱导，但在瞬时转染SF-1的野生型ES细胞中不被cAMP诱导，这表明在类固醇生成发生之前需要诱导下游基因产物。我们证明，破坏SF-1的DNA结合结构域或AF2转录激活结构域的突变体不会赋予ES细胞类固醇生成表型。然而，有趣的是，与VP16激活结构域融合的AF2突变体确实赋予ES细胞类固醇生成表型，但仅在存在一部分配体结合结构域的情况下。这些研究将SF-1在类固醇生成组织中的作用扩展到类固醇生成细胞表型的主要调节因子。
