The orphan nuclear receptor steroidogenic factor-1 regulates the cyclic adenosine 3',5'-monophosphate-mediated transcriptional activation of rat cytochrome P450c17 (17 alpha-hydroxylase/c17-20 lyase).
The orphan nuclear receptor steroidogenic factor-1 regulates the cyclic adenosine 3',5'-monophosphate-mediated transcriptional activation of rat cytochrome P450c17 (17 alpha-hydroxylase/c17-20 lyase).
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DOI：
10.1210/mend.10.2.8825555
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发表时间：
1996-02
影响因子：
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通讯作者：
Peilin Zhang;S. Mellon
中科院分区：
文献类型：
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作者：
Peilin Zhang;S. Mellon
大鼠类固醇细胞色素P450 17 α-羟化酶/c17-20裂解酶（rP 450 c17）基因在类固醇生成组织中受到转录调控。先前的研究表明，位于转录起始位点上游-75和-50碱基对（bp）之间的一个DNA元件介导rP 450 c17的基础和cAMP调节的转录。在凝胶迁移率变动测定和功能测定中使用一系列突变寡核苷酸，现在显示位于-58/-69 bp的12 bp的核心序列对于核蛋白结合和转录激活是必需的。突变体寡核苷酸克隆到含有异源胸苷激酶启动子的荧光素酶报告基因构建体中，转染到小鼠Leydig MA-10和肾上腺皮质Y-1细胞中，得到的结果与凝胶迁移试验的结果一致。突变体，废除结合的核蛋白的DNA废除的基因的基础转录以及对cAMP的反应，而那些突变体，没有废除结合的核蛋白的DNA仍然表现出强大的基础转录以及对cAMP的反应。孤儿核受体类固醇生成因子-1（SF-1）的结合序列与共识结合位点的比较表明，9个碱基中有8个是相同的。然而，来自rP 450 c17的序列在5 '端包括另外三个碱基，先前未证明对SF-1结合重要。在大肠杆菌中表达的重组大鼠SF-1蛋白与该序列结合，并且针对大鼠SF-1产生的抗体消除重组SF-1与来自Y-1和MA-10细胞的核蛋白的结合。这些观察结果表明，rP 450 c17基因的这一区域负责基础转录和cAMP诱导，并与孤儿核受体SF-1结合。进一步显示SF-1可以在体外被蛋白激酶A磷酸化。这种磷酸化发生在丝氨酸和苏氨酸残基处，并导致与rP 450 c17 - 58/-69元件的结合减少。由于SF-1介导cAMP诱导的大鼠P450 c17基因的转录调节，SF-1通过蛋白激酶A的磷酸化可能在转录激活中起调节作用。
The rat steroid cytochrome P450 17 alpha-hydroxylase/c17-20 lyase (rP450c17) gene is transcriptionally regulated in steroidogenic tissues. Previous studies showed that one DNA element located between -75 and -50 base pairs (bp) upstream from the transcriptional initiation site mediated both the basal and cAMP-regulated transcription of rP450c17. Using a series of mutant oligonucleotides in gel mobility shift assays and in functional assays, it is now shown that a core sequence of 12 bp, located at -58/-69 bp, is essential for nuclear protein binding and transcriptional activation. Mutant oligonucleotides cloned into a luciferase reporter gene construct containing a heterologous thymidine kinase promoter, transfected into mouse Leydig MA-10 and adrenocortical Y-1 cells, gave results consistent with those of gel shift assays. Mutants that abolished binding of the nuclear protein to DNA abolished the basal transcription of the gene as well as the responsiveness to cAMP, whereas those mutants that did not abolish binding of the nuclear protein to DNA still showed strong basal transcription as well as responsiveness to cAMP. Comparison of the binding sequence with the consensus binding site for the orphan nuclear receptor steroidogenic factor-1 (SF-1) showed that eight of nine bases were identical. However, the sequence from rP450c17 includes an additional three bases at the 5'-end, not previously demonstrated to be important for SF-1 binding. Recombinant rat SF-1 protein expressed in Escherichia coli binds to this sequence, and antibodies raised against rat SF-1 abolish binding of both recombinant SF-1 and the nuclear protein from Y-1 and MA-10 cells. These observations demonstrate that this region of the rP450c17 gene is responsible for both the basal transcription and cAMP inducibility and is bound by the orphan nuclear receptor SF-1. It is further shown that SF-1 can be phosphorylated in vitro by protein kinase A. This phosphorylation occurs at serine and threonine residues and results in decreased binding to the rP450c17 -58/-69 element. Since SF-1 mediates cAMP-induced transcriptional regulation of the rat P450c17 gene, phosphorylation of SF-1 via protein kinase A is likely to play a regulatory role in transcriptional activation.